Indazole compounds and their uses

ABSTRACT

The present application relates to therapeutic organic compounds, compositions comprising an effective amount of a therapeutic organic compound; and methods for treating and preventing disease comprising administering and effective amount of a therapeutic organic compound to a subject in need thereof.

RELATED APPLICATIONS

This application is a §371 filing based on International Application No.PCT/US2011/025423, filed Feb. 18, 2011, which claims the benefit ofpriority to Provisional U.S. Patent Application Ser. No. 61/314,402filed on Mar. 16, 2010. The contents of these patent applications areincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant #0744413awarded by the National Science Foundation and under R01 grant awardedby the National Institutes of Health. The United States Government hascertain rights in the invention.

BACKGROUND

According to data collected by the American Cancer Society, more than1.43 million people in the United States were diagnosed with cancer in2008. Although earlier diagnoses and improved treatments have allowedfor modest increases in five-year survival rates, the overall mortalityrate per 100,000 people has gone down only 5 percent since 1950 due tothe increased incidence of several types of cancer over the same period(SEER Cancer Statistics Review 1975-2004, NCI “55-Year Trends in U.S.Cancer Death Rates”).

Research directed toward the mechanisms by which cancer cellsproliferate and survive has implicated the deregulation of proteinkinases. Therefore, methods of modulating or inhibiting kinase activity,including the use of small molecule agents, represent a promisingdirection in oncology drug development.

Thus, there remains a need for compounds that inhibit the activity ofone or more protein kinases, as they can be expected to be useful in thetreatment of cancer.

SUMMARY OF THE INVENTION

The invention provides compounds, pharmaceutical compositions comprisingsuch compounds and methods of using such compounds to treat or preventdiseases or disorders associated with abnormal or deregulated kinaseactivity, particularly diseases or disorders that involve abnormalactivation of the Abl, BCR-Abl, EML4-ALK, TEL-ALK, Src or PDGFR kinases.Such diseases include, for example, cancer, e.g. pancreatic cancer,non-small cell lung cancer, gastrointestinal stromal tumor, or chronicmyelogenous leukemia.

Thus, in one aspect provided herein is a compound of Formula I. In oneembodiment, Formula I is represented as Formula II.

In another aspect, provided herein is a method of treating cancer,comprising administering to a subject in need thereof a compound ofFormula I. The cancer can be selected from the group consisting ofadenocarcinoma, multiple myeloma, chronic myelogenous leukemia,pancreatic cancer, non-small cell lung cancer, lung cancer, breastcancer, colon cancer, ovarian cancer, prostate cancer, cervical cancer,uterine cancer, malignant melanoma, non-melanoma skin cancers,gastrointestinal stromal tumors, hematologic tumors, hematologicmalignancies, childhood leukemia, childhood lymphomas, Hodgkin'sdisease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin,acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acutemyelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm,lymphoid neoplasm and cancers associated with AIDS. In anotherembodiment, the cancer is pancreatic cancer or non-small cell lungcancer. In still another embodiment, the cancer is gastrointestinalstromal tumor or chronic myelogenous leukemia. The cancer can beresistant to treatment with Gleevec or Imatinib, whereintreatment-resistance can be due to one or more point-mutations in an Ablkinase, a BCR-Abl kinase domain, a c-kit kinase, a EML4-ALK kinase, aTEL-ALK kinase, an Src kinase or a PDGFR kinase.

In another aspect, provided herein is the use of a compound of Formula Ifor the manufacture of a medicament for treating cancer in a subject.

In yet another aspect, provided herein is a method of inhibiting theactivity of a kinase, comprising utilizing a compound of Formula I. Inone embodiment, the kinase is selected from Abl, Abl (T315I), BCR-Abl,ALK, BLK, CDK5, CDK2, CDK3, CDK7, CDK8, CSF1R, EML4-ALK, FAK, FER, FLT1,FLT3, FLT4, HIPK4, JNK2, KDR, kit, LCK, p38, RET, RIPK1, SLK, TEL-ALK,TIE1, TNK1, TTK or Src comprising utilizing a compound of Formula I.

In another aspect, provided herein is a method of inhibiting theactivity of Abl kinase, BCR-Abl kinase, c-kit kinase, EML4-ALK kinase,TEL-ALK kinase, PDGFRA or PDGFRB kinase, or Src kinase comprisingutilizing a compound of Formula I. In yet another embodiment, providedherein is a method of treating a disease in a subject, wherein thedisease etiology or progression is at least partially mediated by theactivity of Abl kinase, BCR-Abl kinase, c-kit kinase, Src kinase,EML4-ALK kinase, TEL-ALK kinase or PDGFR kinase, comprisingadministering to the subject a compound of Formula I. In still anotherembodiment, provided herein is a method of treating cancer, comprisingadministering to a subject in need thereof a compound of Formula I incombination with a pharmaceutically effective amount of an additionalanti-cancer agent. The additional anti-cancer agent can be imatinib ornilotinib.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the Invention

This invention is directed toward compounds, intermediates thereto andderivatives thereof, as well as pharmaceutical compositions containingthe compounds for use in treatment of protein kinase-associateddisorders. The compounds of the invention or compositions thereof areuseful as inhibitors of the kinase enzymes c-Abl, c-kit, BCR-Abl, PDGFR,EML4-ALK, TEL-ALK, Src and combinations thereof. Furthermore, thecompounds of the invention or compositions thereof can be used in thetreatment of cancer, e.g. pancreatic cancer, non-small cell lung cancer,gastrointestinal stromal tumor, or chronic myelogenous leukemia. Thepresent invention is also directed to methods of inhibiting proteinkinase activity in cells, or for treating, preventing or amelioratingone or more symptoms of cancer using the compounds of the invention orpharmaceutical compositions, in combination with an additionalanti-cancer agent.

In one aspect, the invention provides compounds of the Formula I:

and pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof, wherein

-   -   X is NR, O, S or a bond;        -   wherein R is selected from H and C₁₋₆ alkyl;    -   R^(1a) and R^(1b) are optionally, independently selected from H,        C₁-₆ alkyl, C(O)—C₃-₆ cycloalkyl and C(O)-phenyl,        -   wherein C₁-₆ alkyl, C(O)—C₃-₆ cycloalkyl and C(O)-phenyl can            be independently substituted one or more times with            heterocyclyl, or heterocyclyl-C₁₁-₆ alkyl;    -   R^(A) is selected from H, C₁-₆ alkyl, C₁-₆ alkoxy and QR⁵,        provided that at least one instance of R^(A) is QR⁵;        -   wherein Q is selected from —C(O)—, —C(O)N(H)—, —NHC(O)— and            —N(H)C(O)N(H)—;        -   R⁵ is selected from C₁-₆ alkyl, C₃-₆ cycloalkyl, C₃-₆            cycloheteroalkyl, C₁-₆ alkyloxy, heterocyclyl, or a phenyl            ring;            -   wherein the C₁-₆ alkyl, C₃-₆ cycloalkyl, C₃-₆                cycloheteroalkyl, C₁-₆ alkyloxy, heterocyclyl, or phenyl                ring is optionally, independently substituted one or                more times with: C₁-₆ alkyl, C₂-₆ alkenyl, C₁-₆                heteroalkyl, C₁-₆ monohaloalkyl, C₁-₆ dihaloalkyl, C₁-₆                trihaloalkyl, heterocyclyl, heteroaryl, heteroaryl-C₁-₆                alkyl, or C₁-₆ alkyl-heterocyclyl-C₁-₆ alkyl; and    -   n is selected from 1-5.

In one embodiment of Formula I,

-   -   X is NR, O, S or a bond;        -   wherein R is selected from H and C₁₋₆ alkyl;    -   R^(1a) and R^(1b) are optionally, independently selected from H,        C₁-₆ alkyl, C(O)—C₃-₆ cycloalkyl and C(O)-phenyl,        -   wherein C₁-₆ alkyl, C(O)—C₃-₆ cycloalkyl and C(O)-phenyl can            be independently substituted one or more times with            heterocyclyl, or heterocyclyl-C₁-₆ alkyl;    -   R^(A) is selected from H, C₁-₆ alkyl, C₁-₆ alkoxy and QR⁵,        provided that at least one instance of R^(A) is QR⁵;        -   wherein Q is selected from —C(O)N(H)—, —NHC(O)— and            —N(H)C(O)N(H)—;        -   R⁵ is a phenyl ring that is optionally, independently            substituted one or more times with: C₁-₆ alkyl, C₂-₆            alkenyl, C₁-₆ heteroalkyl, C₁-₆ monohaloalkyl, C₁-₆            dihaloalkyl, C₁-₆ trihaloalkyl, heterocyclyl, heteroaryl,            heteroaryl-C₁-6 alkyl, or C₁-₆ alkyl-heterocyclyl-C₁-₆            alkyl; and    -   n is selected from 1-5.

In one embodiment, Formula I is represented as Formula II:

and pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof, wherein:

-   -   X is NR, O, S or a bond,        -   wherein R is selected from H and C₁₋₆ alkyl;    -   R^(1a) and R^(1b) are optionally, independently selected from H,        C₁-₆ alkyl, C(O)—C₃-₆ cycloalkyl and C(O)-phenyl;        -   wherein the phenyl can be independently substituted one or            more times with, heterocyclyl, or heterocyclyl-C₁-₆ alkyl;    -   R² is selected from H and C₁-₆ alkyl; and    -   R³ and R⁴ are optionally, independently selected from H and QR⁵,        provided that at least one of R³ and R⁴ is QR⁵;        -   wherein Q is selected from —C(O)N(H)—, —NHC(O)— and            —N(H)C(O)N(H)—;        -   R⁵ is a phenyl ring that is optionally, independently            substituted one or more times with: H, C₁-₆ alkyl, C₁-₆            heteroalkyl, C₁-₆ monohaloalkyl, C₁-₆ dihaloalkyl, C₁-₆            trihaloalkyl, heterocyclyl, heteroaryl, heteroaryl-C₁-₆            alkyl, or C₁-₆ alkyl-heterocyclyl-C₁-₆ alkyl;            -   wherein the heterocyclyl moieties can be substituted or                unsubstituted.

In another embodiment of Formula I or II, R^(1a) is C(O)-phenyl and thephenyl is unsubstituted.

In one embodiment of Formula II, R² is selected from H or C₁-₆ alkyl; R³and R⁴ are optionally, independently selected from H and QR⁵, providedthat at least one of R³ and R⁴ is QR⁵; wherein Q is selected from—C(O)N(H)—, —NHC(O)— and —N(H)C(O)N(H)—, and wherein R⁵ is phenyl ringthat is optionally, independently substituted one or more times with: H,C₁-₆ alkyl, C₁-₆ heteroalkyl, C₁-₆ monohaloalkyl, C₁-₆ dihaloalkyl, C₁-₆trihaloalkyl, heterocyclyl, heteroaryl, heteroaryl-C₁-₆ alkyl, or C₁-₆alkyl-heterocyclyl-C₁-₆ alkyl, wherein the heterocyclyl moieties can besubstituted or unsubstituted.

In another embodiment of Formula II, R⁵ is:

wherein R⁶, R⁷, R⁸ and R⁹ are each, independently H, C₁-₆ alkyl, C₁-₆heteroalkyl, C₁-₆ monohaloalkyl, C₁-₆ dihaloalkyl, C₁-₆ trihaloalkyl,heterocyclyl, heteroaryl, heteroaryl-C₁-₆ alkyl, or C₁-₆alkyl-heterocyclyl-C₁-₆ alkyl.

In another embodiment of Formula II, R² is H or C₁-₆ alkyl.

In another embodiment of Formula II, R³ is H or QR⁵.

In another embodiment of Formula II, R⁴ is H or QR⁵.

In another embodiment of Formula II, R⁶ is H.

In another embodiment of Formula II, R⁷ is H, C₁-₆ haloalkyl, orheteroaryl-C₁-₆ alkyl.

In another embodiment of Formula II, R⁸ is H or C₁-₆alkyl-heterocyclyl-C₁-₆ alkyl.

In another embodiment of Formula II, R⁹ is H, C₁-₆ haloalkyl, orheteroaryl-C₁-₆ alkyl.

In another embodiment of Formula II, X is NR, O, S or a bond, wherein Ris selected from H and C₁₋₆ alkyl; R^(1a) and R^(1b) are optionally,independently selected from H, C₁-₆ alkyl, C(O)—C₃-₆ cycloalkyl,C(O)-phenyl and C(O)-phenyl-piperazinyl-C₁-₆ alkyl; R² is H or C₁-₆alkyl; R³ is H or QR⁵; R⁴ is H or QR⁵; wherein R⁵ is:

wherein R⁶ is H; R⁷ is selected from H, C₁-₆ trihaloalkyl, andheterocyclyl-C₁-₆ alkyl; R⁸ is selected from H and C₁-₆alkyl-heterocyclyl-C₁-₆ alkyl; and R⁹ is selected from H, C₁-₆trihaloalkyl, and heterocyclyl-C₁-₆ alkyl.

In another embodiment of Formula II, X is NR, O, or a bond; R is H;R^(1a) is H, CH₃, C(O)-cyclopropyl, C(O)Ph or C(O)Ph-piperazinyl-CH₃;R^(1b) is H; R² is H or CH₃; R³ is H or C(O)N(H)—R⁵; R⁴ is H or QR⁵;wherein R⁵ is:

wherein R⁶ is H; R⁷ is selected from H, CF₃, and imidazolyl-CH₃; R⁸ isselected from H, —CH₂-piperazinyl-CH₃ and —CH₂-piperazinyl-CH₂CH₃; andR⁹ is selected from H, CF₃, and imidazolyl-CH₃.

In another embodiment of Formula II, R⁸ and R⁹ are each, independentlyH, CF₃, 1-ethyl-4-methylpiperazinyl or 4-methyl-1H-imidazolyl,

respectively.

In another embodiment of Formula II, X is NR.

In another embodiment of Formula II, X is O.

In another embodiment of Formula II, X is S.

In another embodiment of Formula II, X is a bond.

In another embodiment of Formula II, R^(1a) is selected from H andC(O)-cyclopropyl; R^(1b) is H; R² is selected from H and C₁₋₆ alkyl; R³and R⁴ are each, independently H or QR⁵; provided that one of R³ and R⁴is H and the other is QR⁵; wherein R⁵ is:

wherein R⁶ is H; R⁷ is CF₃; R⁸ is selected from H and1-ethyl-4-methylpiperazinyl; R⁹ is selected from H and4-methyl-1H-imidazolyl.

In another embodiment of Formula II, R^(1a) is C(O)-cyclopropyl; R^(1b)is H; R² is H; R³ and R⁴ are each, independently H or QR⁵, provided thatone of R³ and R⁴ is H and the other is QR⁵; wherein R⁵ is:

wherein R⁶ is H; R⁷ is CF₃; R⁸ is H or 1-ethyl-4-methylpiperazinyl; R⁹is H or 4-methyl-1H-imidazolyl.

In another embodiment of Formula II, R⁴ is C(O)N(H)—R⁵; wherein R⁵ is:

wherein R⁸ is 1-ethyl-4-methylpiperazinyl and R⁹ is H; or R⁴ isC(O)N(H)—R⁵; wherein R⁵ is:

wherein R⁸ is H and R⁹ is 4-methyl-1H-imidazolyl; or R⁴ is N(H)C(O)—R⁵;wherein R⁵ is:

wherein R⁸ is 1-ethyl-4-methylpiperazinyl and R⁹ is H.

In another embodiment, the compound of Formula I or II is selected fromthe compounds listed in Table A.

Preferred embodiments of Formula I (including pharmaceuticallyacceptable salts thereof, as well as enantiomers, stereoisomers,rotamers, tautomers, diastereomers, atropisomers or racemates thereof)are shown below in Table A and are also considered to be “compounds ofthe invention.” The compounds of the invention are also referred toherein as “protein kinase inhibitors.”

TABLE A Physical Data Compound ¹H NMR 600 MHz and/or MS Number Structure(m/z) 1

¹H NMR 600 MHz (CD₃OD) δ 8.28 (s, 1H), 8.19 (s, 1H), 7.97 (t, 7.8 Hz,2H), 7.93 (d, 8.4 Hz, 1H), 7.88 (d, 8.4 Hz, 1H), 7.73 (s, 1H), 7.63 (t,7.8 Hz, 1H), 7.56 (t, 7.8 Hz, 1H), 7.48 (d, 8.4 Hz, 1H), 7.44 (d, 7.8Hz, 1H), 1.95 (m, 1H), 1.04 (s, 2H), 0.94 (m, 2H), MS m/z: 465.15 (M +1). 2

¹H NMR 600 MHz (DMSO-d6) δ 11.52 (s, 1H), 10.59 (s, 1H), 8.26 (s, 1H),8.21 (d, 1.8 Hz, 1H), 8.05 (dd, 8.4, 1.8 Hz, 1H), 7.92 (m, 2H), 7.76 (d,8.4 Hz, 1H), 7.71 (d, 8.4 Hz, 1H), 7.61 (m, 1H), 7.54 (s, 1H), 7.28 (dd,8.4, 1.8 Hz, 1H), 5.98 (q, 4.8 Hz, 1H), 3.55 (s, 2H), 2.86 (d, 4.8 Hz,3H), 2.3-2.5 (m, 10H), 0.97 (t, 7.2 Hz, 3H). MS m/z: 537.25 (M + 1). 3

MS m/z: 627.26 (M + 1). 4

MS m/z: 591.26 (M + 1). 6

MS m/z: 545.18 (M + 1). 7

MS m/z: 434 (M + 1). 8

MS m/z: 458 (M + 1). 9

MS m/z: 523 (M + 1). 10

MS m/z: 361 (M + 1). 11

MS m/z: 351 (M + 1). 12

MS m/z: 335 (M + 1). 13

MS m/z: 605 (M + 1). 14

MS m/z: 537 (M + 1). 15

MS m/z: 479 (M + 1). 16

MS m/z: 559 (M + 1). 17

MS m/z: 523.23 (M + 1). 18

MS m/z: 491.17 (M + 1). 19

MS m/z: 591.26 (M + 1). 20

MS m/z: 523.23 (M + 1). 21

MS m/z: 606.27 (M + 1). 22

MS m/z: 361.16 (M + 1). 23

MS m/z: 591.26 (M + 1). 24

MS m/z: 606.27 (M + 1). 25

MS m/z: 494 (M + 1). 26

MS m/z: 622 (M + 1). 27

MS m/z: 484 (M + 1). 28

MS m/z: 599 (M + 1).Methods of Treatment

Compounds of the present invention are useful for the treatment ofprotein kinase-associated disorders.

As used herein, the term “protein kinase-associated disorder” includesdisorders and states (e.g., a disease state) that are associated withthe activity of a protein kinase. Non-limiting examples of proteinkinase-associated disorders include abnormal cell proliferation,including protein kinase-associated cancers, viral infections, fungalinfections, autoimmune diseases and neurodegenerative disorders.

Non-limiting examples of protein-kinase associated disorders includeproliferative diseases, such as viral infections, auto-immune diseases,fungal disease, cancer, psoriasis, vascular smooth cell proliferationassociated with atherosclerosis, pulmonary fibrosis, arthritisglomerulonephritis, chronic inflammation, neurodegenerative disorders,such as Alzheimer's disease, and post-surgical stenosis and restenosis.Protein kinase-associated disorders also include diseases related toabnormal cell proliferation, including, but not limited to, cancers ofthe head and neck, breast, ovary, cervix, prostate, testis, esophagus,stomach, skin, lung, bone, colon, pancreas, thyroid, biliary passages,buccal cavity and pharynx (oral), larynx, lip, tongue, mouth, smallintestine, colon-rectum, large intestine, rectum, prostate, brain andcentral nervous system, glioblastoma, neuroblastoma, keratoacanthoma,epidermoid carcinoma, large cell carcinoma, adenocarcinoma, adenoma,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma,seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma, kidneycarcinoma, myeloid disorders, lymphoid disorders, Hodgkin's, hairycells, and leukemia.

Protein kinase-associated disorders also include diseases associatedwith apoptosis, including, but not limited to, cancer, viral infections,autoimmune diseases and neurodegenerative disorders.

Examples of protein kinase-associated cancers include carcinomas,hematopoietic tumors of lymphoid lineage, hematopoietic tumors ofmyeloid lineage, tumors of mesenchymal origin, tumors of the central andperipheral nervous system, melanoma, seminoma, teratocarcinoma,osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroidfollicular cancer and Kaposi's sarcoma.

Additional, non-limiting examples of protein-kinase associated disordersinclude tumor angiogenesis and metastasis. Non-limiting examples ofprotein-kinase associated disorders also include vascular smooth muscleproliferation associated with atherosclerosis, postsurgical vascularstenosis and restenosis, and endometriosis.

Non-limiting examples of protein-kinase associated disorders includeviral infections in a patient in need thereof, wherein the viralinfections include, but are not limited to, HIV, human papilloma virus,herpes virus, poxyirus, Epstein-Barr virus, Sindbis virus andadenovirus.

Further non-limiting examples of protein-kinase associated disordersinclude those associated with infectious agents, including yeast, fungi,protozoan parasites such as Plasitiodium falciparum, and DNA and RNAviruses.

Compounds of the present invention are useful for the treatment ofcancer, wherein the cancer is selected from the group consisting ofmultiple myeloma, chronic myelogenous leukemia, pancreatic cancer,non-small cell lung cancer, lung cancer, breast cancer, colon cancer,ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skincancers, gastrointestinal stromal tumors, hematologic tumors,hematologic malignancies, childhood leukemia, childhood lymphomas,multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic origin,lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acutelymphoblastic leukemia, acute myelocytic leukemia, chronic myelocyticleukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associatedwith AIDS.

In another embodiment, compounds of the present invention are used formodulating the activity of a protein kinase, including, but not limitedto, protein kinases selected from the group consisting of Abl, ATK,BCR-Abl, Blk, Brk, Btk, BRAF, c-fms, e-kit, c-met, c-src, CDK, cRafl,CSFIR, CSK, EGFR, EML4-ALK, ErbB2, ErbB3, ErbB4, ERK, DDR-1, Fak, fes,FGFRI, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK-4, flt-1, flt-3, flt-4, Fps,Frk, Fyn, GSK, Gst-Flkl, Hck, Her-2, Her-4, HIPK-1, IGF-IR, INS-R, Jak,JNK, KDR, Lck, LOK, Lyn, MEK, p38, panHER, PDGFR, PLK, PKC, PYK2, Raf,Rho, ros, SRC, TEL-ALK, TRK, TYK2, UL97, VEGFR, Yes, Zap70, Aurora-A,GSK3-alpha, HIPK1, HIPK2, HIP3, 1RAK1, JNK1, JNK2, JNK3, TRKB, CAMKII,CK1, CK2, RAF, GSK3Beta, MAPK1, MKK4, MKK7, MST2, NEK2, AAK1, PKCalpha,PKD, RET, RIPK2, ROCK-II, and TIE2

As used herein, the term “modulating” or “modulation” refers to thealteration of the catalytic activity of a protein kinase. In particular,modulating refers to the activation of the catalytic activity of aprotein kinase, preferably the activation or inhibition of the catalyticactivity of a protein kinase, depending on the concentration of thecompound or salt to which the protein kinase is exposed or, morepreferably, the inhibition of the catalytic activity of a proteinkinase. The term “catalytic activity” as used herein refers to the rateof phosphorylation of tyrosine, serine or threonine under the influence,direct or indirect, of a protein kinase.

The three main classes that pharmacological inhibitors of kinaseactivity are categorized by are (1) Type I, or “DFG-in” ATP competitiveinhibitors, which directly compete with ATP in the ATP binding site(i.e. dual Src, ABL inhibitor dasatinib, (2) Type II, or “DFG-out” ATPcompetitive inhibitors, which, in addition to binding the ATP bindingsite also engage an adjacent hydrophobic binding site that is onlyaccessible when the kinase is in an inactivated configuration (i.e. theactivation loop is oriented in a conformation that would block substratebinding) (i.e. imatinib, nilotinib), and (3) non-ATP competitiveinhibitors that binds at sites outside the ATP binding site that affectthe activity of the kinase (i.e. GNF-2).

Second generation Abl inhibitors, such as dasatinib, and nilotinib, arehighly active against imatinib-resistant leukemia. Both agents aresignificantly more potent against Bcr-Abl than imatinib, and are activeagainst many imatinib-resistant Bcr-Abl mutants. However, neither agentis able to override imatinib resistance due to the mutation of athreonine to an isoleucine at residue 315 (T315I, the “gatekeeper”position). This highly prevalent and highly imatinib-resistant mutationis centrally located in the nucleotide binding cleft of Abl. Bothdasatinib and nilotinib make a hydrogen bonding interaction to theside-chain hydroxyl group of T315, which is resistant to these compoundsdue to direct steric intrusion of the isobutyl side chain and a loss inthe middle of the ATP-cleft of a hydrogen-bonding interaction.

In addition to BCR-Abl T315I, other gatekeeper residues that play anintegral role in imatinib-resistant disease include c-kit-T670I, whichis associated with imatinib-resistant gastrointestinal stromal tumorcharacterized by early metastasis and shorter progression-free survival;this mutation substantially modifies the binding pocket of c-Kit, andoccurs only under the selective pressure of imatinib therapy,PDGFRA-T674M/I, which is found in the FIP1LI-PDGFRA kinase domain andgives rise to imatinib resistance in idiopathic hypereosinophilicsyndrome (HES), and PDGFRB-T681M/I.

The compounds of the invention are type II class kinase inhibitors thattraverse the gatekeeper position in a manner that accommodates aminoacid side chains of a variety of sizes.

The above-listed protein kinases may exhibit one or more pointmutations, including, but not limited to mutations of the hinge region,mutations of the P-loop, and mutations of the A-loop.

In a preferred embodiment, the protein kinase is selected from the groupconsisting of mutated or non-mutated Abl, mutated or non-mutated c-kit,mutated or non-mutated BCR-Abl, mutated or non-mutated PDGFR, mutated ornon-mutated Src and any combination thereof. In a particularly preferredembodiment, the protein kinase is selected from the group consisting ofmutated or non-mutated c-kit, mutated or non-mutated BCR-Abl, mutated ornon-mutated PDGFR, mutated or non-mutated Src, mutated or non-mutatedTEL-ALK and mutated or non-mutated EML4-ALK.

In one embodiment, a compound of the present invention is characterizedas an inhibitor of a combination of protein kinases, e.g., BCR-Abland/or c-kit and/or PDGFR.

In certain embodiments, a compound of the present invention is used forprotein kinase-associated diseases, and/or as an inhibitor of any one ormore protein kinases. It is envisioned that a use can involve theinhibition one or more isoforms of the protein kinase.

The efficacy of therapeutic kinase inhibitors such as imatinib,dasatinib and nilotinib is typically correlated with their affinitytoward one or more kinase targets that are associated with a particulardisease state. Point-mutations, which can occur naturally or under theselective pressure of chemotherapy, can decrease the affinity of thechemotherapeutic for its kinase target, thereby conferring resistance tothese therapies.

The compounds of the invention are also inhibitors of mutated ornon-mutated forms of the kinase enzymes Abl, BCR-Abl, c-kit PDGFR,EML4-ALK, 1EL-ALK or Src, which are implicated in certain disease statesrelated to cancer, e.g. pancreatic cancer, non-small cell lung cancer,gastrointestinal stromal tumor, or chronic myelogenous leukemia. BCR-Ablactivates a number of cell cycle-controlling proteins and enzymes,speeding up cell division and inhibiting DNA repair, thus resulting ingenomic instability and, potentially, blast crisis in CML. Aberrantactivation of c-kit is observed in most gastrointestinal stromal tumors,while the effects of PDGFR include cell proliferation and angiogenesis.

Without being bound by theory, it is believed that inhibition of thekinase enzymes Abl, BCR-Abl, c-kit, PDGFR, EML4-ALK, TEL-ALK and Srcwill promote apoptosis, inhibit cancer cell proliferation and inhibittumor growth.

The present invention also includes treatment of one or more symptoms ofcancer, e.g. pancreatic cancer, non-small cell lung cancer,gastrointestinal stromal tumor, or chronic myelogenous leukemia, as wellas protein kinase-associated disorders such as those described above,but the invention is not intended to be limited to the manner by whichthe compound performs its intended function of treatment of a disease.The present invention includes treatment of diseases described herein inany manner that allows treatment to occur.

In certain embodiments, the compounds of the invention are used alone orin combination with other therapeutic agents, e.g. imatinib, nilotinibor dasatinib.

In another embodiment, the invention provides a pharmaceuticalcomposition of any of the compounds of the present invention. In arelated embodiment, the invention provides a pharmaceutical compositionof any of the compounds of the present invention and a pharmaceuticallyacceptable carrier or excipient. In certain embodiments, the inventionincludes the compounds as novel chemical entities.

In other embodiments, the present invention provides a method forinhibiting the activity of a protein kinase. The method includescontacting a cell with any of the compounds of the present invention. Ina related embodiment, the method further provides that the compound ispresent in an amount effective to selectively inhibit the activity of aprotein kinase.

Additionally, a method of the invention includes administering to asubject an effective amount of a protein kinase-modulating compound ofthe invention, e.g., protein kinase-modulating compounds of Formula I,as well as Table A (including pharmaceutically acceptable salts thereof,as well as enantiomers, stereoisomers, rotamers, tautomers,diastereomers, atropisomers or racemates thereof).

In other embodiments, the present invention provides a use of any of thecompounds of the invention for manufacture of a medicament to treatcancer.

In other embodiments, the invention provides a method of manufacture ofa medicament, including formulating any of the compounds of the presentinvention for treatment of a subject.

One embodiment provided herein is a method of treating pancreaticcancer, comprising administering compound 6 to a subject in needthereof, such that the pancreatic cancer is treated.

Another embodiment provided herein is a method of treating non-smallcell lung cancer, comprising administering compound 6 to a subject inneed thereof, such that the non-small cell lung cancer is treated.

In yet another embodiment provided herein is a method of treatinggastrointestinal stromal tumor, comprising administering compound 6 to asubject in need thereof, such that the gastrointestinal stromal tumor istreated.

In still another embodiment embodiment provided herein is a method oftreating chronic myelogenous leukemia, comprising administering compound6 to a subject in need thereof, such that the chronic myelogenousleukemia is treated.

One other embodiment provided herein is a method of treating pancreaticcancer, comprising administering compound 19 to a subject in needthereof, such that the pancreatic cancer is treated.

Another embodiment provided herein is a method of treating non-smallcell lung cancer, comprising administering compound 19 to a subject inneed thereof, such that the non-small cell lung cancer is treated.

In yet another embodiment provided herein is a method of treatinggastrointestinal stromal tumor, comprising administering compound 19 toa subject in need thereof, such that the gastrointestinal stromal tumoris treated.

In still another embodiment embodiment provided herein is a method oftreating chronic myelogenous leukemia, comprising administering compound19 to a subject in need thereof, such that the chronic myelogenousleukemia is treated.

One embodiment, provided herein is the use of compound 6 for themanufacture of a medicament for the treatment of pancreatic cancer in asubject in need thereof.

In another embodiment, provided herein is the use of compound 6 for themanufacture of a medicament for the treatment of non-small cell lungcancer in a subject in need thereof.

In yet another embodiment, provided herein is the use of compound 6 forthe manufacture of a medicament for the treatment of gastrointestinalstromal tumor in a subject in need thereof.

In still another embodiment, provided herein is the use of compound 6for the manufacture of a medicament for the treatment of chronicmyelogenous leukemia in a subject in need thereof.

One other embodiment, provided herein is the use of compound 19 for themanufacture of a medicament for the treatment of pancreatic cancer in asubject in need thereof.

In another embodiment, provided herein is the use of compound 19 for themanufacture of a medicament for the treatment of non-small cell lungcancer in a subject in need thereof.

In yet another embodiment, provided herein is the use of compound 19 forthe manufacture of a medicament for the treatment of gastrointestinalstromal tumor in a subject in need thereof.

In still another embodiment, provided herein is the use of compound 19for the manufacture of a medicament for the treatment of chronicmyelogenous leukemia in a subject in need thereof.

Definitions

The term “treat,” “treated,” “treating” or “treatment” includes thediminishment or alleviation of at least one symptom associated or causedby the state, disorder or disease being treated. In certain embodiments,the treatment comprises the induction of a protein kinase-associateddisorder, followed by the activation of the compound of the invention,which would in turn diminish or alleviate at least one symptomassociated or caused by the protein kinase-associated disorder beingtreated. For example, treatment can be diminishment of one or severalsymptoms of a disorder or complete eradication of a disorder.

The term “use” includes any one or more of the following embodiments ofthe invention, respectively: the use in the treatment of proteinkinase-associated disorders; the use for the manufacture ofpharmaceutical compositions for use in the treatment of these diseases,e.g., in the manufacture of a medicament; methods of use of compounds ofthe invention in the treatment of these diseases; pharmaceuticalpreparations having compounds of the invention for the treatment ofthese diseases; and compounds of the invention for use in the treatmentof these diseases; as appropriate and expedient, if not statedotherwise. In particular, diseases to be treated and are thus preferredfor use of a compound of the present invention are selected from cancer,e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinalstromal tumor, or chronic myelogenous leukemia, or inflammation, cardiachypertrophy, and HIV infection, as well as those diseases that depend onthe activity of protein kinases. The term “use” further includesembodiments of compositions herein that bind to a protein kinasesufficiently to serve as tracers or labels, so that when coupled to afluor or tag, or made radioactive, can be used as a research reagent oras a diagnostic or an imaging agent.

The term “subject” is intended to include organisms, e.g., prokaryotesand eukaryotes, which are capable of suffering from or afflicted with adisease, disorder or condition associated with the activity of a proteinkinase. Examples of subjects include mammals, e.g., humans, dogs, cows,horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenicnon-human animals. In certain embodiments, the subject is a human, e.g.,a human suffering from, at risk of suffering from, or potentiallycapable of suffering from cancer, e.g. pancreatic cancer, non-small celllung cancer, gastrointestinal stromal tumor, or chronic myelogenousleukemia, or inflammation, cardiac hypertrophy, and HIV infection, andother diseases or conditions described herein (e.g., a proteinkinase-associated disorder). In another embodiment, the subject is acell.

The language “protein kinase-modulating compound,” “modulator of proteinkinase” or “protein kinase inhibitor” refers to compounds that modulate,e.g., inhibit, or otherwise alter, the activity of a protein kinase.Examples of protein kinase-modulating compounds include compounds of theinvention, i.e., Formula I, as well as the compounds of Table A(including pharmaceutically acceptable salts thereof, as well asenantiomers, stereoisomers, rotamers, tautomers, diastereomers,atropisomers or racemates thereof).

As used herein, the term “alkyl” refers to a fully saturated branched orunbranched hydrocarbon moiety. Preferably the alkyl comprises 1 to 20carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 10 carbonatoms, 1 to 7 carbon atoms, or 1 to 4 carbon atoms. Representativeexamples of alkyl include, but are not limited to, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,n-decyl and the like. Furthermore, the expression “C_(x)-C_(y)-alkyl”,wherein x is 1-5 and y is 2-10 indicates a particular alkyl group(straight- or branched-chain) of a particular range of carbons. Forexample, the expression C₁-C₄-alkyl includes, but is not limited to,methyl, ethyl, propyl, butyl, isopropyl, tert-butyl and isobutyl.

The term “alkenyl,” alone or in combination refers to a straight-chain,cyclic or branched hydrocarbon residue comprising at least one olefinicbond and the indicated number of carbon atoms. Preferred alkenyl groupshave up to 8, preferably up to 6, particularly preferred up to 4 carbonatoms. Examples of alkenyl groups are ethenyl, 1-propenyl, 2-propenyl,isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, isobutenyl,1-cyclohexenyl, 1-cyclopentenyl.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength to the alkyls described above, but which contain at least onetriple bond. For example, the term “alkynyl” includes straight-chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynylgroups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. Theterm alkynyl further includes alkynyl groups that include oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkynyl group has 6 or fewer carbon atoms in its backbone(e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). The termC₂-C₆ includes alkynyl groups containing 2 to 6 carbon atoms.

As used herein, the term “cycloalkyl” refers to saturated or unsaturatedmonocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbonatoms, preferably 3-9, or 3-7 carbon atoms. Exemplary monocyclichydrocarbon groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl andthe like. Exemplary bicyclic hydrocarbon groups include bornyl, indyl,hexahydroindyl, tetrahydronaphthyl, decahydronaphthyl,bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl,6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl,bicyclo[2.2.2]octyl and the like. Exemplary tricyclic hydrocarbon groupsinclude adamantyl and the like.

The term “cycloalkenyl” refers to a partially unsaturated cyclichydrocarbon group containing 1 to 3 rings and 4 to 8 carbons per ring.Exemplary groups include cyclobutenyl, cyclopentenyl, and cyclohexenyl.The term “cycloalkenyl” also includes bicyclic and tricyclic groups inwhich at least one of the rings is a partially unsaturated,carbon-containing ring and the second or third ring may be carbocyclicor heterocyclic, provided that the point of attachment is to thecycloalkenyl group.

“Alkoxy” refers to those alkyl groups, having from 1 to 10 carbon atoms,attached to the remainder of the molecule via an oxygen atom. Alkoxygroups with 1-8 carbon atoms are preferred. The alkyl portion of analkoxy may be linear, cyclic, or branched, or a combination thereof.Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, butoxy,cyclopentyloxy, and the like. An alkoxy group can also be represented bythe following formula: —OR^(i), where R^(i) is the “alkyl portion” of analkoxy group.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, consisting of the stated number of carbon atomsand from one to five heteroatoms, more preferably from one to threeheteroatoms, selected from the group consisting of O, N, Si and S, andwherein the nitrogen and sulfur atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroalkyl groupis attached to the remainder of the molecule through a carbon atom or aheteroatom.

The term “alkylcarbonyl” refers to a group having the formula—C(O)—R^(ii), wherein R^(ii) is an alkyl group as defined above andwherein the total number of carbon atoms refers to the combined alkyland carbonyl moieties. An “alkylcarbonyl” group can be attached to theremainder of the molecule via an alkyl group (i.e., -alkyl-C(O)—R^(ii)).

The term “alkoxycarbonyl” refers to a group having the formula—C(O)O—R^(iii), wherein R^(iii) is an alkyl group as defined above andwherein the total number of carbon atoms refers to the combined alkyland carbonyl moieties. An “alkoxycarbonyl” group can be attached to theremainder of the molecule via an alkyl group (i.e.,-alkyl-C(O)O—R^(iii)).

The term “heteroalkylcarbonyl” refers to a group having the formula—C(O)R^(iv), wherein R^(iv) is a heteroalkyl group as defined above andwherein the total number of carbon atoms refers to the combined alkyland carbonyl moieties. A “heteroalkylcarbonyl” group can be attached tothe remainder of the molecule via an alkyl or heteroalkyl group (i.e.,-alkyl-C(O)O—R^(iv) or -heteroalkyl-C(O)O—R^(iv)).

The term “aryl” includes aromatic monocyclic or multicyclic e.g.,tricyclic, bicyclic, hydrocarbon ring systems consisting only ofhydrogen and carbon and containing from six to nineteen carbon atoms, orsix to ten carbon atoms, where the ring systems may be partiallysaturated. Aryl groups include, but are not limited to, groups such asphenyl, tolyl, xylyl, anthryl, naphthyl and phenanthryl. Aryl groups canalso be fused or bridged with alicyclic or heterocyclic rings which arenot aromatic so as to form a polycycle (e.g., tetralin).

The term “heteroaryl,” as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition ofheterocycle below, “heteroaryl” is also understood to include theN-oxide derivative of any nitrogen-containing heteroaryl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively.

The term “heterocycle” or “heterocyclyl” refers to a five-member toten-member, fully saturated or partially unsaturated nonaromaticheterocylic groups containing at least one heteroatom such as O, S or N.The most frequent examples are piperidinyl, morpholinyl, piperazinyl,pyrrolidinyl or pirazinyl. Attachment of a heterocyclyl substituent canoccur via a carbon atom or via a heteroatom.

Moreover, the alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkoxy, aryl,heteroaryl, and heterocycle groups described above can be“unsubstituted” or “substituted.” The term “substituted” is intended todescribe moieties having substituents replacing a hydrogen on one ormore atoms, e.g. C, O or N, of a molecule. Such substituents canindependently include, for example, one or more of the following:straight or branched alkyl (preferably C₁-C₅), cycloalkyl (preferablyC₃-C₈), alkoxy (preferably C₁₋C₆), thioalkyl (preferably C₁-C₆), alkenyl(preferably C₂-C₆), alkynyl (preferably C₂₋C₆), heterocyclic,carbocyclic, aryl (e.g., phenyl), aryloxy (e.g., phenoxy), aralkyl(e.g., benzyl), aryloxyalkyl (e.g., phenyloxyalkyl), arylacetamidoyl,alkylaryl, heteroaralkyl, alkylcarbonyl and arylcarbonyl or other suchacyl group, heteroarylcarbonyl, or heteroaryl group, (CR′R″)₀₋₃NR′R″(e.g., —NH₂), (CR′R″)₀₋₃CN (e.g., —CN), —NO₂, halogen (e.g., —F, —Cl,—Br, or —I), (CR′R″)₀₋₃C(halogen)₃ (e.g., —CF₃), (CR′R″)₀₋₃CH(halogen)₂,(CR′R″)₀₋₃CH₂(halogen), (CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃(CNH)NR′R″,(CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO, (CR′R″)₀₋₃O(CR′R″)₀₋₃H,(CR′R″)₀₋₃S(O)₀₋₃R′ (e.g., —SO₃H, —OSO₃H), (CR′R″)₀₋₃O(CR′R″)₀₋₃H (e.g.,—CH₂OCH₃ and —OCH₃), (CR′R″)₀₋₃S(CR′R″)₀₋₃H (e.g., —SH and —SCH₃),(CR′R″)₀₋₃OH (e.g., —OH), (CR′R″)₀₋₃COR′, (CR′R″)₀₋₃ (substituted orunsubstituted phenyl), (CR′R″)₀₋₃(C₃-C₈ cycloalkyl), (CR′R″)₀₋₃CO₂R′(e.g., —CO₂H), or (CR′R″)₀₋₃OR′ group, or the side chain of anynaturally occurring amino acid; wherein R′ and R″ are each independentlyhydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, or aryl group.

The term “amine” or “amino” should be understood as being broadlyapplied to both a molecule, or a moiety or functional group, asgenerally understood in the art, and may be primary, secondary, ortertiary. The term “amine” or “amino” includes compounds where anitrogen atom is covalently bonded to at least one carbon, hydrogen orheteroatom. The terms include, for example, but are not limited to,“alkyl amino,” “arylamino,” “diarylamino,” “alkylarylamino,”“alkylaminoaryl,” “arylaminoalkyl,” “alkaminoalkyl,” “amide,” “amido,”and “aminocarbonyl.” The term “alkyl amino” comprises groups andcompounds wherein the nitrogen is bound to at least one additional alkylgroup. The term “dialkyl amino” includes groups wherein the nitrogenatom is bound to at least two additional alkyl groups. The term“arylamino” and “diarylamino” include groups wherein the nitrogen isbound to at least one or two aryl groups, respectively. The term“alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl” refers to anamino group which is bound to at least one alkyl group and at least onearyl group. The term “alkaminoalkyl” refers to an alkyl, alkenyl, oralkynyl group bound to a nitrogen atom which is also bound to an alkylgroup.

The term “amide,” “amido” or “aminocarbonyl” includes compounds ormoieties which contain a nitrogen atom which is bound to the carbon of acarbonyl or a thiocarbonyl group. The term includes “alkaminocarbonyl”or “alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl oralkynyl groups bound to an amino group bound to a carbonyl group. Itincludes arylaminocarbonyl and arylcarbonylamino groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarbonyl,” “alkenylaminocarbonyl,” “alkynylaminocarbonyl,”“arylaminocarbonyl,” “alkylcarbonylamino,” “alkenylcarbonylamino,”“alkynylcarbonylamino,” and “arylcarbonylamino” are included in term“amide.”Amides also include urea groups (aminocarbonylamino) andcarbamates (oxycarbonylamino).

In a particular embodiment of the invention, the term “amine” or “amino”refers to substituents of the formulas N(R⁸)R⁹, CH₂N(R⁸)R⁹ andCH(CH₃)N(R⁸)R⁹, wherein R⁸ and R⁹ are each, independently, selected fromthe group consisting of H and (C₁-C₄-alkyl)₀₋₁G, wherein G is selectedfrom the group consisting of COOH, H, PO_(S)H, SO₃H, Br, Cl, F,O—C₁₋₄-alkyl, S—C₁₋₄-alkyl, aryl, C(O)OC₁-C₆-alkyl,C(O)C₁-C₄-alkyl-COOH, C(O)C₁-C₄-alkyl and C(O)-aryl.

The description of the disclosure herein should be construed incongruity with the laws and principals of chemical bonding. For example,it may be necessary to remove a hydrogen atom in order accommodate asubstitutent at any given location. Furthermore, it is to be understoodthat definitions of the variables (i.e., “R groups”), as well as thebond locations of the generic formulae of the invention (e.g., FormulasI or II), will be consistent with the laws of chemical bonding known inthe art. It is also to be understood that all of the compounds of theinvention described above will further include bonds between adjacentatoms and/or hydrogens as required to satisfy the valence of each atom.That is, bonds and/or hydrogen atoms are added to provide the followingnumber of total bonds to each of the following types of atoms: carbon:four bonds; nitrogen: three bonds; oxygen: two bonds; and sulfur:two-six bonds.

The compounds of this invention may include asymmetric carbon atoms. Itis to be understood accordingly that the isomers arising from suchasymmetry (e.g., all enantiomers, stereoisomers, rotamers, tautomers,diastereomers, or racemates) are included within the scope of thisinvention. Such isomers can be obtained in substantially pure form byclassical separation techniques and by stereochemically controlledsynthesis. Furthermore, the structures and other compounds and moietiesdiscussed in this application also include all tautomers thereof.Compounds described herein may be obtained through art recognizedsynthesis strategies.

It will also be noted that the substituents of some of the compounds ofthis invention include isomeric cyclic structures. It is to beunderstood accordingly that constitutional isomers of particularsubstituents are included within the scope of this invention, unlessindicated otherwise. For example, the term “tetrazole” includestetrazole, 2H-tetrazole, 3H-tetrazole, 4H-tetrazole and 5H-tetrazole.

Isotopes

The present invention includes all pharmaceutically acceptableisotopically-labeled compounds of the invention, i.e. compounds ofFormulas (I) and (II), wherein one or more atoms are replaced by atomshaving the same atomic number, but an atomic mass or mass numberdifferent from the atomic mass or mass number usually found in nature.

Examples of isotopes suitable for inclusion in the compounds of theinvention comprises isotopes of hydrogen, such as ²H and ³H, carbon,such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F,iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, oxygen,such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulphur, such as³⁵S.

Certain isotopically-labelled compounds of Formulas (I) and (II), forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies. The radioactive isotopestritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularly useful forthis purpose in view of their ease of incorporation and ready means ofdetection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy.

Isotopically-labeled compounds of Formulas (I) and (II) can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described in the accompanying Examplesand Preparations using an appropriate isotopically-labeled reagents inplace of the non-labeled reagent previously employed.

Combinations

The compounds of this invention are also useful in combination withknown anti-cancer agents. Such known anti-cancer agents include thefollowing: estrogen receptor modulators, androgen receptor modulators,retinoid receptor modulators, cytotoxic agents, antiproliferativeagents, prenyl-protein transferase inhibitors, HMG-CoA reductaseinhibitors, HIV protease inhibitors, reverse transcriptase inhibitors,and other angiogenesis inhibitors.

“Estrogen receptor modulators” refers to compounds, which interfere orinhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phe-nyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds, which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,.alpha.-difluoromethylornithine, ILX23-7553,trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic agents” refer to compounds which cause cell death primarilyby interfering directly with the cell's functioning or inhibit orinterfere with cell myosis, including alkylating agents, tumor necrosisfactors, intercalators, microtubulin inhibitors, and topoisomeraseinhibitors.

Examples of cytotoxic agents include, but are not limited to,tirapazimine, sertenef, cachectin, ifosfamide, tasonermin, lonidamine,carboplatin, doxorubicin, altretamine, prednimustine, dibromodulcitol,ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomnide,heptaplatin, estramustine, improsulfan tosilate, trofosfainide,nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin,profiromycin, cisplatin, irofulven, dexifosfamide,cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine,glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamiine(-chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

Examples of microtubulin inhibitors include paclitaxel, vindesinesulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol,rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin,RPR109881, BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]indolizino[1,2b]quinoline-10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin,BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide,sobuzoxane, 2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazo-le-1-carboxamide,asulacrine, (5a, 5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydrox-y-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d-)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridiniu-m,6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-py-razolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylaminol]-7-methoxy-9-oxo-9H-thioxanthen4-ylmethyl-]formamide,N-(2-(dimethylamino)ethyl)acridine4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino4-oxo4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(—S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)4-formyl-6-methoxy-14-oxa-1,11-diazatetra-cyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine, and3-amninopyridine-2-carboxaldehyde thiosemicarbazone.

“Antiproliferative agents” also includes monoclonal antibodies to growthfactors, other than those listed under “angiogenesis inhibitors”, suchas trastuzumab, and tumor suppressor genes, such as p53, which can bedelivered via recombinant virus-mediated gene transfer (see U.S. Pat.No. 6,069,134, for example).

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Compounds which haveinhibitory activity for HMG-CoA reductase can be readily identified byusing assays well-known in the art. For example, see the assaysdescribed or cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131at pp. 30-33. The terms “HMG-CoA reductase inhibitor” and “inhibitor ofHMG-CoA reductase” have the same meaning when used herein.

Examples of HMG-CoA reductase inhibitors that may be used include, butare not limited to lovastatin, simvastatin, pravastatin, fluvastatin,atorvastatin and cerivastatin. The structural formulae of these andadditional HMG-CoA reductase inhibitors that may be used in the instantmethods are described at page 87 of M. Yalpani, “Cholesterol LoweringDrugs”, Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos.4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as usedherein includes all pharmaceutically acceptable lactone and open-acidforms (i.e., where the lactone ring is opened to form the free acid) aswell as salt and ester forms of compounds which have HMG-CoA reductaseinhibitory activity, and therefore the use of such salts, esters,open-acid and lactone forms is included within the scope of thisinvention.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase). Examples of prenyl-protein transferase inhibiting compoundsinclude (+/−)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(−)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]4-(3-chloro-phenyl)-1-methyl-2(1H)-quinolinone,(+)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,5(S)-n-butyl-1-(2,3-dimethylphenyl)4-[1-(4-cyanobenzyl)-5-imidazolylmethy-1]-2-piperazinone,(S)-1-(3-chlorophenyl)4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-5-[2-(ethanesulfonyl)methyl)-2-piperazinone,5(S)-n-Butyl-1-(2-methylphenyl)4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-2-methyl-5-imidazolylmethyl]-2-pi-perazinone,1-(2,2-diphenylethyl)-3-[N-(1-(4-cyanobenzyl)-1H-imidazol-5-ylethyl)carba-moyl]piperidine,4-{5-[4-hydroxymethyl4-(4-chloropyridin-2-ylmethyl)-piperidine-1-ylmethyl-]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{5-[4-hydroxymethyl-4-(3-chlo-robenzyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-pyridin-1-yl)benzyl]-3H-imidazol4-ylmethyl}benzonitrile-,4-{3-[4-(5-chloro-2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol4-ylm-ethyl}benzonitrile,4-{3-[4-(2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol4-ylmethyl}benz-onitrile,4-[3-(2-oxo-1-phenyl-1,2-dihydropyridin4-ylmethyl)-3H-imidazol4-ylmethyl}benzonitrile,18,19-dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-1H-imidazo[4,3-c][1,11,4]dioxaazacyclo-nonadecine-9-carbonitrile,(+/−)-19,20-dihydro-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriaza-cyclooctadecine-9-carbonitrile,19,20-dihydro-19-oxo-5H,17H-18,21-ethano-6,10:12,16-dimetheno-22H-imidazo-[3,4-h][1,8,11,14]oxatriazacycloeicosine-9-carbonitrile,and(+/−)-19,20-dihydro-3-methyl-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-me-theno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxa-triazacyclooctadecine-9-carbonitrile.

Other examples of prenyl-protein transferase inhibitors can be found inthe following publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510,5,589,485, 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European J. ofCancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

Examples of HIV protease inhibitors include amprenavir, abacavir,CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, tipranavir,ritonavir, saquinavir, ABT-378, AG 1776, and BMS-232,632. Examples ofreverse transcriptase inhibitors include delaviridine, efavirenz,GS-840, HB Y097, lamivudine, nevirapine, AZT, 3TC, ddC, and ddI.

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR20), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, interferon-.alpha.,interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors,including nonsteroidal anti-inflammatories (NSAIDs) like aspirin andibuprofen as well as selective cyclooxygenase-2 inhibitors likecelecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69,p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec.,Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin,Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107(1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol.57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med.,Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),carboxyamidotriazole, combretastatin A-4, squalamnine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-I, angiotensin II antagonists (see Fernandez et al., J. Lab.Clin. Med. 105:141-145 (1985)), and antibodies to VEGF. (see, NatureBiotechnology, Vol. 17, pp. 963-968 (October 1999); Kim et al., Nature,362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Assays

The inhibition of protein kinase activity by the compounds of theinvention may be measured using a number of assays available in the art.Examples of such assays are described in the Exemplification sectionbelow.

Pharmaceutical Compositions

The compounds of the present invention are suitable as active agents inpharmaceutical compositions that are efficacious particularly fortreating protein kinase-associated disorders and cancer, e.g. pancreaticcancer, non-small cell lung cancer, gastrointestinal stromal tumor, orchronic myelogenous leukemia. The pharmaceutical composition in variousembodiments has a pharmaceutically effective amount of the presentactive agent along with other pharmaceutically acceptable excipients,carriers, fillers, diluents and the like.

The language “pharmaceutically effective amount” of the compound is thatamount necessary or sufficient to treat or prevent a proteinkinase-associated disorder, e.g. prevent the various morphological andsomatic symptoms of a protein kinase-associated disorder, and/or adisease or condition described herein. In an example, an effectiveamount of a compound of the invention is the amount sufficient to treata protein kinase-associated disorder in a subject. The effective amountcan vary depending on such factors as the size and weight of thesubject, the type of illness, or the particular compound of theinvention. For example, the choice of the compound of the invention canaffect what constitutes an “effective amount.” One of ordinary skill inthe art would be able to study the factors contained herein and make thedetermination regarding the effective amount of the compounds of theinvention without undue experimentation.

The regimen of administration can affect what constitutes an effectiveamount. A compound of the invention can be administered to the subjecteither prior to or after the onset of a protein kinase-associateddisorder. Further, several divided dosages, as well as staggered dosagescan be administered daily or sequentially, or the dose can becontinuously infused, or can be a bolus injection. Further, the dosagesof the compound(s) of the invention can be proportionally increased ordecreased as indicated by the exigencies of the therapeutic orprophylactic situation.

The phrase “pharmaceutically acceptable amount” of a compound of thepresent invention refers to an amount of a compound of the presentinvention that will elicit the biological or medical response of asubject, for example, reduction or inhibition of an enzyme or a proteinactivity, or ameliorate symptoms, alleviate conditions, slow or delaydisease progression, or prevent a disease, etc. In one non-limitingembodiment, the phrase “pharmaceutically acceptable amount” refers tothe amount of a compound of the present invention that, whenadministered to a subject, is effective to (1) at least partiallyalleviate, inhibit, prevent and/or ameliorate a condition, or a disorderor a disease (i) mediated by kinase enzymes c-Abl, BCR-Abl, c-kit, Src,EML4-ALK, TEL-ALK and/or PDGFR, or (ii) associated with kinase enzymesc-Abl, BCR-Abl, c-kit, Src, EML4-ALK, TEL-ALK and/or PDGFR activity, or(iii) characterized by abnormal activity of kinase enzymes c-Abl,BCR-Abl, c-kit, Src, EML4-ALK, TEL-ALK and/or PDGFR; or (2) reduce orinhibit the activity of kinase enzymes c-Abl, BCR-Abl, c-kit, Src,EML4-ALK, TEL-ALK and/or PDGFR; or (3) reduce or inhibit the expressionof kinase enzymes c-Abl, BCR-Abl, c-kit, Src, EML4-ALK, TEL-ALK and/orPDGFR. In another non-limiting embodiment, the phrase “pharmaceuticallyacceptable amount” refers to the amount of a compound of the presentinvention that, when administered to a subject, is effective to at leastpartially alleviate, inhibit, prevent and/or ameliorate cancer, e.g.pancreatic cancer, non-small cell lung cancer, gastrointestinal stromaltumor, or chronic myelogenous leukemia. In still another non-limitingembodiment, the term “pharmaceutically acceptable amount” refers to theamount of a compound of the present invention that, when administered toa cell, or a tissue, or a non-cellular biological material, or a medium,is effective to at least partially reduce or inhibit the activity ofkinase enzymes c-Abl, BCR-Abl, c-kit, Src, EML4-ALK, TEL-ALK and/orPDGFR; or at least partially reduce or inhibit the expression of kinaseenzymes c-Abl, BCR-Abl, c-kit, Src, EML4-ALK, TEL-ALK and/or PDGFR.

The acceptable amount can vary depending on such factors as the size andweight of the subject, the type of illness, or the particular organiccompound. For example, the choice of the organic compound can affectwhat constitutes an “acceptable amount.” One of ordinary skill in theart would be able to study the aforementioned factors and make thedetermination regarding the acceptable amount of the organic compoundwithout undue experimentation.

Compounds of the invention may be used in the treatment of states,disorders or diseases as described herein, or for the manufacture ofpharmaceutical compositions for use in the treatment of these diseases.Methods of use of compounds of the present invention in the treatment ofthese diseases, or pharmaceutical preparations having compounds of thepresent invention for the treatment of these diseases.

The language “pharmaceutical composition” includes preparations suitablefor administration to mammals, e.g., humans. When the compounds of thepresent invention are administered as pharmaceuticals to mammals, e.g.,humans, they can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical, buccal, sublingual, rectal, vaginal and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient that can be combined with acarrier material to produce a single dosage form will generally be thatamount of the compound that produces a therapeutic effect. Generally,out of one hundred per cent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably fromabout 5 per cent to about 70 per cent, most preferably from about 10 percent to about 30 per cent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc., administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral and/or IV administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous and subcutaneousdoses of the compounds of this invention for a patient, when used forthe indicated analgesic effects, will range from about 0.0001 to about100 mg per kilogram of body weight per day, more preferably from about0.01 to about 50 mg per kg per day, and still more preferably from about1.0 to about 100 mg per kg per day. An effective amount is that amounttreats a protein kinase-associated disorder.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

Synthetic Procedure

Compounds of the present invention are prepared from commonly availablecompounds using procedures known to those skilled in the art, includingany one or more of the following conditions without limitation:

Acid addition salts of the compounds of the invention are most suitablyformed from pharmaceutically acceptable acids, and include for examplethose formed with inorganic acids e.g. hydrochloric, hydrobromic,sulphuric or phosphoric acids and organic acids e.g. succinic, malaeic,acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g.oxalates can be used for example in the isolation of the compounds ofthe invention, for laboratory use, or for subsequent conversion to apharmaceutically acceptable acid addition salt. Also included within thescope of the invention are solvates and hydrates of the invention.

The conversion of a given compound salt to a desired compound salt isachieved by applying standard techniques, in which an aqueous solutionof the given salt is treated with a solution of base e.g. sodiumcarbonate or potassium hydroxide, to liberate the free base which isthen extracted into an appropriate solvent, such as ether. The free baseis then separated from the aqueous portion, dried, and treated with therequisite acid to give the desired salt.

In vivo hydrolyzable esters or amides of certain compounds of theinvention can be formed by treating those compounds having a freehydroxy or amino functionality with the acid chloride of the desiredester in the presence of a base in an inert solvent such as methylenechloride or chloroform. Suitable bases include triethylamine orpyridine. Conversely, compounds of the invention having a free carboxygroup can be esterified using standard conditions which can includeactivation followed by treatment with the desired alcohol in thepresence of a suitable base.

Examples of pharmaceutically acceptable addition salts include, withoutlimitation, the non-toxic inorganic and organic acid addition salts suchas the hydrochloride derived from hydrochloric acid, the hydrobromidederived from hydrobromic acid, the nitrate derived from nitric acid, theperchlorate derived from perchloric acid, the phosphate derived fromphosphoric acid, the sulphate derived from sulphuric acid, the formatederived from formic acid, the acetate derived from acetic acid, theaconate derived from aconitic acid, the ascorbate derived from ascorbicacid, the benzenesulphonate derived from benzensulphonic acid, thebenzoate derived from benzoic acid, the cinnamate derived from cinnamicacid, the citrate derived from citric acid, the embonate derived fromembonic acid, the enantate derived from enanthic acid, the fumaratederived from fumaric acid, the glutamate derived from glutamic acid, theglycolate derived from glycolic acid, the lactate derived from lacticacid, the maleate derived from maleic acid, the malonate derived frommalonic acid, the mandelate derived from mandelic acid, themethanesulphonate derived from methane sulphonic acid, thenaphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, thephthalate derived from phthalic acid, the salicylate derived fromsalicylic acid, the sorbate derived from sorbic acid, the stearatederived from stearic acid, the succinate derived from succinic acid, thetartrate derived from tartaric acid, the toluene-p-sulphonate derivedfrom p-toluene sulphonic acid, and the like. Particularly preferredsalts are sodium, lysine and arginine salts of the compounds of theinvention. Such salts can be formed by procedures well known anddescribed in the art.

Other acids such as oxalic acid, which can not be consideredpharmaceutically acceptable, can be useful in the preparation of saltsuseful as intermediates in obtaining a chemical compound of theinvention and its pharmaceutically acceptable acid addition salt.

Metal salts of a chemical compound of the invention include alkali metalsalts, such as the sodium salt of a chemical compound of the inventioncontaining a carboxy group.

Mixtures of isomers obtainable according to the invention can beseparated in a manner known per se into the individual isomers;diastereoisomers can be separated, for example, by partitioning betweenpolyphasic solvent mixtures, recrystallisation and/or chromatographicseparation, for example over silica gel or by, e.g., medium pressureliquid chromatography over a reversed phase column, and racemates can beseparated, for example, by the formation of salts with optically puresalt-forming reagents and separation of the mixture of diastereoisomersso obtainable, for example by means of fractional crystallisation, or bychromatography over optically active column materials.

Intermediates and final products can be worked up and/or purifiedaccording to standard methods, e.g., using chromatographic methods,distribution methods, (re-) crystallization, and the like.

At all stages of the reactions, mixtures of isomers that are formed canbe separated into the individual isomers, for example diastereoisomersor enantiomers, or into any desired mixtures of isomers, for exampleracemates or mixtures of diastereoisomers, for example analogously tothe methods described in Science of Synthesis: Houben-Weyl Methods ofMolecular Transformation. Georg Thieme Verlag, Stuttgart, Germany, 2005.

The solvents from which those solvents that are suitable for anyparticular reaction may be selected include those mentioned specificallyor, for example, water, esters, such as lower alkyl-lower alkanoates,for example ethyl acetate, ethers, such as aliphatic ethers, for examplediethyl ether, or cyclic ethers, for example tetrahydrofuran or dioxane,liquid aromatic hydrocarbons, such as benzene or toluene, alcohols, suchas methanol, ethanol or 1- or 2-propanol, nitriles, such asacetonitrile, halogenated hydrocarbons, such as methylene chloride orchloroform, acid amides, such as dimethylformamide or dimethylacetamide, bases, such as heterocyclic nitrogen bases, for examplepyridine or N-methylpyrrolidin-2-one, carboxylic acid anhydrides, suchas lower alkanoic acid anhydrides, for example acetic anhydride, cyclic,linear or branched hydrocarbons, such as cyclohexane, hexane orisopentane, or mixtures of those solvents, for example aqueoussolutions, unless otherwise indicated in the description of theprocesses. Such solvent mixtures may also be used in working up, forexample by chromatography or partitioning.

The compounds, including their salts, may also be obtained in the formof hydrates, or their crystals can, for example, include the solventused for crystallization. Different crystalline forms may be present.

The invention relates also to those forms of the process in which acompound obtainable as an intermediate at any stage of the process isused as starting material and the remaining process steps are carriedout, or in which a starting material is formed under the reactionconditions or is used in the form of a derivative, for example in aprotected form or in the form of a salt, or a compound obtainable by theprocess according to the invention is produced under the processconditions and processed further in situ.

Prodrugs

This invention also encompasses pharmaceutical compositions containing,and methods of treating protein kinase-associated disorders throughadministering, pharmaceutically acceptable prodrugs of compounds of thecompounds of the invention. For example, compounds of the inventionhaving free amino, amido, hydroxy or carboxylic groups can be convertedinto prodrugs. Prodrugs include compounds wherein an amino acid residue,or a polypeptide chain of two or more (e.g., two, three or four) aminoacid residues is covalently joined through an amide or ester bond to afree amino, hydroxy or carboxylic acid group of compounds of theinvention. The amino acid residues include but are not limited to the 20naturally occurring amino acids commonly designated by three lettersymbols and also includes 4-hydroxyproline, hydroxylysine, demosine,isodemosine, 3-methylhistidine, norvalin, beta-alanine,gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithineand methionine sulfone. Additional types of prodrugs are alsoencompassed. For instance, free carboxyl groups can be derivatized asamides or alkyl esters. Free hydroxy groups may be derivatized usinggroups including but not limited to hemisuccinates, phosphate esters,dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlinedin Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs ofhydroxy and amino groups are also included, as are carbonate prodrugs,sulfonate esters and sulfate esters of hydroxy groups. Derivatization ofhydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein theacyl group may be an alkyl ester, optionally substituted with groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities, or where the acyl group is an amino acid ester asdescribed above, are also encompassed. Prodrugs of this type aredescribed in J. Med. Chem. 1996, 39, 10. Free amines can also bederivatized as amides, sulfonamides or phosphonamides. All of theseprodrug moieties may incorporate groups including but not limited toether, amine and carboxylic acid functionalities.

Any reference to a compound of the present invention is therefore to beunderstood as referring also to the corresponding pro-drugs of thecompound of the present invention, as appropriate and expedient.

Kits

Advantageously, the present invention also provides kits for use by aconsumer for treating disease. The kits comprise a) a pharmaceuticalcomposition comprising an antibiotic and a pharmaceutically acceptablecarrier, vehicle or diluent; and, optionally, b) instructions describinga method of using the pharmaceutical composition for treating thespecific disease. The instructions may also indicate that the kit is fortreating disease while substantially reducing the concomitant liabilityof adverse effects associated with antibiotic administration.

A “kit” as used in the instant application includes a container forcontaining the separate unit dosage forms such as a divided bottle or adivided foil packet. The container can be in any conventional shape orform as known in the art which is made of a pharmaceutically acceptablematerial, for example a paper or cardboard box, a glass or plasticbottle or jar, a re-sealable bag (for example, to hold a “refill” oftablets for placement into a different container), or a blister packwith individual doses for pressing out of the pack according to atherapeutic schedule. The container employed can depend on the exactdosage form involved, for example a conventional cardboard box would notgenerally be used to hold a liquid suspension. It is feasible that morethan one container can be used together in a single package to market asingle dosage form. For example, tablets may be contained in a bottlewhich is in turn contained within a box.

An example of such a kit is a so-called blister pack. Blister packs arewell known in the packaging industry and are being widely used for thepackaging of pharmaceutical unit dosage forms (tablets, capsules, andthe like). Blister packs generally consist of a sheet of relativelystiff material covered with a foil of a preferably transparent plasticmaterial. During the packaging process, recesses are formed in theplastic foil. The recesses have the size and shape of individual tabletsor capsules to be packed or may have the size and shape to accommodatemultiple tablets and/or capsules to be packed. Next, the tablets orcapsules are placed in the recesses accordingly and the sheet ofrelatively stiff material is sealed against the plastic foil at the faceof the foil which is opposite from the direction in which the recesseswere formed. As a result, the tablets or capsules are individuallysealed or collectively sealed, as desired, in the recesses between theplastic foil and the sheet. Preferably the strength of the sheet is suchthat the tablets or capsules can be removed from the blister pack bymanually applying pressure on the recesses whereby an opening is formedin the sheet at the place of the recess. The tablet or capsule can thenbe removed via said opening.

It may be desirable to provide a written memory aid, where the writtenmemory aid is of the type containing information and/or instructions forthe physician, pharmacist or subject, e.g., in the form of numbers nextto the tablets or capsules whereby the numbers correspond with the daysof the regimen which the tablets or capsules so specified should beingested or a card which contains the same type of information. Anotherexample of such a memory aid is a calendar printed on the card e.g., asfollows “First Week, Monday, Tuesday,” . . . etc. . . . “Second Week,Monday, Tuesday, . . . ” etc. Other variations of memory aids will bereadily apparent. A “daily dose” can be a single tablet or capsule orseveral tablets or capsules to be taken on a given day.

Another specific embodiment of a kit is a dispenser designed to dispensethe daily doses one at a time. Preferably, the dispenser is equippedwith a memory-aid, so as to further facilitate compliance with theregimen. An example of such a memory-aid is a mechanical counter, whichindicates the number of daily doses that, has been dispensed. Anotherexample of such a memory-aid is a battery-powered micro-chip memorycoupled with a liquid crystal readout, or audible reminder signal which,for example, reads out the date that the last daily dose has been takenand/or reminds one when the next dose is to be taken.

EXEMPLIFICATION OF THE INVENTION

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology and immunology, which are within the skill of theart.

General Synthesis Processes

General Synthesis Methods

Non-limiting general synthesis procedures are depicted in Scheme 1 andScheme 2. As shown in Scheme 1, step I involves condensation of thefluoronitrile compound with hydrazine, using conditions well-known inthe art (e.g., using a solvent such as n-butanol at a temperature suchas 130° C.) to afford a 2-aminoindazole product. Steps II. and III.involve optional alkylation or acylation of the indicated nitrogenatoms, with reactants comprising R, R^(1a) and R^(1b) groups, usingconditions well-known in the art (e.g., using a solvent such as pyridineat a temperature such as 0° C.). Step IV. involves the coupling ofreactants bearing Y and W groups respectively, wherein Y and W arefunctional groups known to those of skill in the art to undergo suchreactions, under conditions well-known in the art (e.g., using a metalcatalyst such as Pd(dppf)Cl₂, a base such as Na₂CO₃, and a solvent suchas dioxane/water at a temperature such as 100° C.).

As shown in Scheme 2, step I. involves condensation of the fluoronitrilecompound with hydrazine, using conditions well-known in the art (e.g.,using a solvent such as n-butanol at a temperature such as 130° C.) toafford a 2-aminoindazole product. Steps II. and III. involve optionalalkylation or acylation of the indicated nitrogen atoms, with reactantscomprising R, R^(1a) and R^(1b) groups, using conditions well-known inthe art (e.g., using a solvent such as pyridine at a temperature such as0° C.). Step IV. involves the coupling of reactants bearing Y and Zgroups respectively, wherein Y and Z are functional groups known tothose of skill in the art to undergo such reactions, under conditionswell-known in the art (e.g., using a metal catalyst such as Pd₂(dba)₃, aligand such as X-Phos, a base such as K₂CO₃, and a solvent such ast-butanol).

All starting materials, building blocks, reagents, acids, bases,dehydrating agents, solvents, and catalysts utilized to synthesis thecompounds of the present invention are either commercially available orcan be produced by organic synthesis methods known to one of ordinaryskill in the art (Houben-Weyl 4th Ed. 1952, Methods of OrganicSynthesis, Thieme, Volume 21). Further, the compounds of the presentinvention can be produced by organic synthesis methods known to one ofordinary skill in the art as shown in the following examples.

Referring to the examples that follow, compounds of the preferredembodiments were synthesized using the methods described herein, orother methods, which are known in the art.

Synthesis Examples

6-Bromo-1H-indazol-3-amine

The reaction mixture of 4-bromo-2-fluorobenzonitrile (10 g, 50 mmol),hydrazine hydrate (7.85 ml, 250 mmol) in n-butanol (60 mL) was heated at130° C. for overnight. After cooling down to room temperature, theprecipitated crystalline solid was filtered off and washed three timeswith ethyl acetate (15 mL each). The product was dried in vacuo (10.3g).

¹H NMR 600 MHz (DMSO-d6) δ 11.48 (s, 1H); 7.62 (d, 8.4 Hz, 1H), 7.40 (d,1.2 Hz, 1H), 6.99 (dd, 8.4, 1.2 Hz, 1H), 5.43 (s, 2H). MS m/z: 211.97(M+1).

N-(6-bromo-1H-indazol-3-yl)cyclopropanecarboxamide

To a solution of 6-bromo-1H-indazol-3-amine (4.24 g, 20 mmol) inpyridine (100 mL) was added cyclopropanecarbonyl chloride (1.83 mL, 20mmol) dropwise at 0° C. The reaction mixture was stirred at thistemperature for 4 hours. Once the reaction was completed, the solventwas removed in vacuo. The residue was dissolved in DMF, and water wasadded dropwise. The precipitated solid was filtered off and washed threetimes with hexanes (15 ml each). The product was dried in vacuo and usedwithout further purification (4.3 g).

¹H NMR 600 MHz (DMSO-d6) δ 12.71 (s, 1H), 10.72 (s, 1H), 7.74 (d, 8.4Hz, 1H), 7.62 (d, 1.2 Hz, 1H), 7.14 (dd, 8.4, 1.2 Hz, 1H), 1.90 (m, 1H),0.82 (m, 4H). MS m/z: 280.0 (M+1).

Ethyl 3-(3-(cyclopropanecarboxamido)-1H-indazol-6-yl)benzoate

To a solution of N-(6-bromo-1H-indazol-3-yl)cyclopropanecarboxamide (560mg, 2.0 mmol) in dioxane (20 mL) and 1N sodium carbonate solution (8 mL)were added 3-(ethoxycarbonyl)phenylboronic acid (388 mg, 2.0 mmol) andPd(dppf)Cl₂ (103 mg, 0.2 mmol). After the reaction was stirred at 100°C. for 4 hours and cooled to room temperature, the mixture was filteredthrough Celite and washed by ethyl acetate. The combined organicsolution was washed by brine, dried with sodium sulfate and concentratedin vacuo. The crude residue was purified by flash chromatography with20:1 (v/v) methylene chloride-methanol to afford the title product (560mg).

¹H NMR 600 MHz (DMSO-d6) δ 12.70 (s, 1H), 10.69 (s, 1H), 8.21 (s, 1H),7.99 (d, 7.8 Hz, 1H), 7.96 (d, 7.8 Hz, 1H), 7.88 (d, 8.4 Hz, 1H), 7.63(m, 2H), 7.34 (d, 8.4 Hz, 1H), 4.35 (q, 7.2 Hz, 2H), 1.94 (m, 1H), 1.34(t, 7.2 Hz, 3H), 0.8-0.9 (m, 4H). MS m/z: 350.14 (M+1).

3-(3-(Cyclopropanecarboxamido)-1H-indazol-6-yl)-N-(3-(trifluoromethyl)phenylbenzamide

To a solution of ethyl3-(3-(cyclopropanecarboxamido)-1H-indazol-6-yl)benzoate (42 mg, 0.12mmol) in THF/MeOH/H₂O was added lithium hydroxide monohydrate (50 mg,1.2 mmol). The reaction mixture was stirred at room temperature forovernight. Once the reaction was completed, the reaction solvent wasremoved to one third and the pH was adjusted to about 6. The formedprecipitate was filtered, dried in vacuo and used without furtherpurification. To a solution of above acid in DMSO were added3-(trifluoromethyl)benzenamine (25 μL, 0.20 mmol),O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (57 mg, 0.15 mmol) and DIEA (52 μL, 0.30mmol). The reaction mixture was stirred at room temperature forovernight and purified by HPLC to afford the title product (10 mg).

¹H NMR 600 MHz (CD₃OD) δ 8.28 (s, 1H), 8.19 (s, 1H), 7.97 (t, 7.8 Hz,2H), 7.93 (d, 8.4 Hz, 1H), 7.88 (d, 8.4 Hz, 1H), 7.73 (s, 1H), 7.63 (t,7.8 Hz, 1H), 7.56 (t, 7.8 Hz, 1H), 7.48 (d, 8.4 Hz, 1H), 7.44 (d, 7.8Hz, 1H), 1.95 (m, 1H), 1.04 (s, 2H), 0.94 (m, 2H). MS m/z: 465.15 (M+1).

Phenyl-6-bromo-1H-indazol-3-yl carbamate

To a solution of 6-bromo-1H-indazol-3-amine (1.27 g, 6.0 mmol) inpyridine (24 mL) was added phenyl chloroformate (0.83 mL, 6.6 mmol)dropwise at 0° C. After stirring at this temperature for 4 hours, thereaction was quenched by water. After the solvent was removed, theresidue was dissolved in ethyl acetate and washed by 1N HCl and brine.The organic layer was dried with sodium sulfate and concentrated invacuo. The crude residue was purified by flash chromatography with 30:70(v/v) ethyl acetate-hexanes to afford the title product (0.80 g).

¹H NMR 600 MHz (DMSO-d6) δ 12.82 (s, 1H), 10.53 (br, 1H), 7.77 (d, 8.4Hz, 1H), 7.68 (d, 1.2 Hz, 1H), 7.40 (m, 2H), 7.23 (m, 3H), 7.20 (dd,8.4, 1.2 Hz, 1H). MS m/z: 332.0 (M+1).

6-Bromo-N-methyl-1H-indazol-3-amine

To a solution of phenyl 6-bromo-1H-indazol-3-ylcarbamate (0.80 g, 2.42mmol) in 1,4-dioxane (40 mL) was added lithium aluminum hydride (2 Msolution in THF, 2.42 mL, 2.42 mmol) dropwise at room temperature. Afterstirring at 100° C. for 4 hours, the reaction was quenched by 0.2 mLwater, followed by 0.2 mL 15% NaOH and 0.6 mL water. The reactionmixture was then filtered and the solid was washed by ethyl acetate. Thecombined organic layer was washed by brine, dried with sodium sulfateand concentrated in vacuo. The crude residue was purified by flashchromatography with 50:50 (v/v) ethyl acetate-hexanes to afford thetitle product (0.29 g).

¹H NMR 600 MHz (DMSO-d6) δ 11.50 (s, 1H), 7.58 (d, 8.4 Hz, 1H), 7.41 (d,1.2 Hz, 1H), 7.00 (dd, 8.4, 1.2 Hz, 1H), 6.02 (q, 4.8 Hz, 1H), 2.82 (d,4.8 Hz, 3H). MS m/z: 226.0 (M+1).

N-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-3-amine

To a solution of 6-bromo-N-methyl-1H-indazol-3-amine (158 mg, 0.7 mmol)in DMF (20 mL) were added bis(pinacolato)diboron (196 mg, 0.77 mmol),KOAc (206 mg, 2.1 mmol), dppf (39 mg, 0.07 mmol), and Pd(dppf)Cl₂ (57mg, 0.07 mmol). After the reaction was stirred at 100° C. for 4 hoursand cooled to room temperature, the mixture was filtered through Celiteand washed by ethyl acetate. The combined organic solution was washed bysodium bicarbonate solution and brine, dried with sodium sulfate andconcentrated in vacuo. The crude residue was purified by flashchromatography with 50:50 (v/v) ethyl acetate-hexanes to afford thetitle product (175 mg).

¹H NMR 600 MHz (DMSO-d6) δ 11.41 (s, 1H), 7.62 (d, 7.8 Hz, 1H), 7.55 (s,1H), 7.14 (d, 7.8 Hz, 1H), 5.91 (q, 4.8 Hz, 1H), 2.83 (d, 4.8 Hz, 3H),1.29 (s, 12H). MS m/z: 274.16 (M+1).

N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-(3-(methylamino)-1H-indazol-6-yl)benzamide

To a solution ofN-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-3-amine(14 mg, 0.05 mmol) in dioxane (1.25 mL) and 1N sodium carbonate solution(0.25 mL) were addedN-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-iodobenzamide(26 mg, 0.05 mmol) and Pd(dppf)Cl₂ (4 mg, 5 μmol). After the reactionwas stirred at 100° C. for 4 hours and cooled to room temperature, themixture was filtered through Celite and washed by ethyl acetate. Thecombined organic solution was washed by brine, dried with sodium sulfateand concentrated in vacuo. The crude residue was purified by HPLC toafford the title product (4.9 mg).

¹H NMR 600 MHz (DMSO-d6) δ 11.52 (s, 1H), 10.59 (s, 1H), 8.26 (s, 1H),8.21 (d, 1.8 Hz, 1H), 8.05 (dd, 8.4, 1.8 Hz, 1H), 7.92 (m, 2H), 7.76 (d,8.4 Hz, 1H), 7.71 (d, 8.4 Hz, 1H), 7.61 (m, 1H), 7.54 (s, 1H), 7.28 (dd,8.4, 1.8 Hz, 1H), 5.98 (q, 4.8 Hz, 1H), 3.55 (s, 2H), 2.86 (d, 4.8 Hz,3H), 2.3-2.5 (m, 10H), 0.97 (t, 7.2 Hz, 3H). MS m/z: 537.25 (M+1).

Ethyl 3-(3-amino-1H-indazol-6-yl)benzoate

To a solution of 6-bromo-1H-indazol-3-amine (2.1 g, 10.0 mmol) indioxane (100 mL) and sodium carbonate (1N, 40 mL) were added3-(ethoxycarbonyl)phenylboronic acid (1.94 g, 10.0 mmol) and Pd(dppf)Cl₂(816 mg, 1.0 mmol). After the reaction was stirred at 100° C. for 4hours and cooled to room temperature, the mixture was filtered throughCelite and washed by ethyl acetate. The combined organic solution waswashed by brine, dried with sodium sulfate and concentrated in vacuo.The crude residue was purified by flash chromatography with 50:1 (v/v)methylene chloride-methanol to afford the title product (1.46 g), MSm/z: 282.11 (M+1).

Ethyl3-(3-(4-(4-methylpiperazin-1-yl)benzamido)-1H-indazol-6-yl)benzoate

To a stirred suspension of 4-(4-methylpiperazin-1-yl)benzoic acid(220.27 mg, 1.0 mmol) in 5 mL of dichloromethane and 20 μL of DMF wasadded oxalyl chloride (87 μL, 1.0 mmol) at 0° C. After stirring at roomtemperature for 2 hours, the reaction was concentrated by rotavapor. Theresidue was dried under vacuum pump and the crude was used in next stepwithout further purification.

To a stirred solution of ethyl 3-(3-amino-1H-indazol-6-yl)benzoate (160mg) in 5 mL of pyridine was added the suspension of the above acidchloride in 4 mL of DMF at 0° C. Then the reaction was warmed up to roomtemperature gradually and stirred overnight. After the reaction wascomplete as monitored by reverse phase analytical liquid-chromatographyelectrospray mass spectrometry (LC-MS), 100 mL of dichloromethane wasadded and the resulting mixture was washed with brine. The organic layerwas dried over anhydrous sodium sulfate and concentrated. The residuewas purified by silica-gel chromatography with dichloromethane: 3.5 Nammonia methanol solution (50:1 v/v) to give the title compound (126mg), MS m/z: 484.22 (M+1).

3-(3-(4-(4-methylpiperazin-1-yl)benzamido)-1H-indazol-6-yl)-N-(3-(trifluoromethyl)phenyl)benzamide

To a solution of ethyl3-(3-(cyclopropanecarboxamido)-1H-indazol-6-yl)benzoate (121 mg, 0.25mmol) in THF/MeOH (2.0 mL/2.0 mL) was added 2.0 mL of 1.0 N lithiumhydroxide aqueous solution. The reaction mixture was stirred at roomtemperature. After the reaction was complete as monitored by LC-MS, thesolvent was removed to one third and the pH was adjusted to about 6 by1N HCl solution. The resulted precipitate was filtered, dried in vacuoand used without further purification.

To a solution of the above acid (23 mg, 0.05 mmol) in 1.0 mL of DMSO wasadded 3-(trifluoromethyl)benzenamine (25 μL, 0.20 mmol),O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (38 mg, 0.10 mmol) and DIEA (52 μL, 0.30mmol). The reaction mixture was stirred at room temperature overnightand purified by reverse-phase prep-HPLC using a water (0.05%TFA)/acetonitrile (0.05% TFA) gradient to afford the title compound asthe TFA salt (8 mg), MS m/z: 599.23 (M+1).

N-(6-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)cyclopropanecarboxamide

To a solution of 6-bromo-1H-indazol-3-amine (560 mg, 2.0 mmol) in DMF(20 mL) was added sodium hydride (160 mg, 60% in mineral oil) at 0° C.After 30 mins, (2-(chloromethoxy)ethyl)trimethylsilane (0.39 mL, 2.2mmol) was added dropwise. The reaction was stirred at 0° C. until it wascomplete as monitored by LC-MS. The resulting mixture was poured intoice-water and extracted by ethyl acetate. The organic layer was washedby brine, dried with anhydrous sodium sulfate and concentrated in vacuo.The crude residue was purified by flash chromatography with 1:5 (v/v)ethyl acetate-hexane afford the title product (548 mg, 67%), MS (ESI)m/z 411 (M+H)⁺.

3-(3-(cyclopropanecarboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-6-ylamino)-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide

To a solution ofN-(6-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)cyclo-propanecarboxamide(41 mg, 0.1 mmol) in t-BuOH (1.5 mL) were added3-amino-4-methyl-N-(3-(trifluoromethyl)-phenyl)benzamide (29 mg, 0.1mmol) and K₂CO₃ (42 mg, 0.3 mmol). The reaction mixture was degassed for10 minutes. To a mixture were added Pd₂(dba)₃ (6.0 mg) and t-Bu-X-phos(4 mg). The reaction mixture was heated at 100° C. until it was completeas monitored by LC-MS. Then the resulting mixture was filtered through apad of celite and eluted with dichloromethane. The to solvent wasremoved by rotavapor and the crude residue was purified by flashchromatography with methylene chloride-methanol to afford the titleproduct (25 mg, 40%), MS (ESI) m/z 624 (M+H)⁺.

3-(3-(cyclopropanecarboxamido)-1H-indazol-6-ylamino)-4-methyl-N-(3-(trifluoro-methyl)phenyl)benzamide

To a solution of3-(3-(cyclopropanecarboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-6-ylamino)-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide(25 mg) in CH₂Cl₂ (1 mL) was added TFA (0.25 mL) at room temperature.The reaction mixture was stirred for 2 hours and the organic solvent wasconcentrated under reduced pressure. To a solution of the resultingmixture in THF (0.5 mL) and MeOH (0.5 mL) was added LiOH aqueoussolution (1.0 M, 1.0 mL). The resulting mixture was stirred for another2 hours at room temperature. The organic solvent was removed underreduced pressure and the aqueous layer was extracted with ethyl acetate.The organic extracts were washed with brine, dried over MgSO₄ andconcentrated under reduced pressure. The crude product was purified byPrep HPLC and acetonitrile was removed under reduced pressure. Theremained water was freeze-dried to afford TFA salt of title compound (16mg, 66%), MS (ESI) m/z 494 (M+H)⁺.

Chemical Compounds and Biologic Reagents

All compounds are initially dissolved in DMSO to make 10 mM stocksolutions, and then are serially diluted to obtain final concentrationsfor in vitro experiments.

Cell Lines and Cell Culture

Ba/F3.p210 cells are obtained by transfecting the IL-3-dependent marinehematopoietic Ba/F3 cell line with a pGD vector containing p210BCR-ABL(B2A2) cDNA (Daley and Baltimore, 1988; Sattler et al., 1996; Okuda etal., 1996). Murine hematopoietic 32D cells are transduced withretrovirus to express p210 Bcr-ABL (32D.p210 cells) (Matulonis et al.,1993); the 32D-T315I cell line is transfected by electroporation withthe imatinib resistant BCR-ABL construct (pCI-neo Mammalian ExpressionVector; Promega (#E1841) harboring the point mutation T315I (Weisberg etal., 2005). Ba/F3 cells are stably transfected by electroporation withimatinib-resistant BCR-ABL constructs (pCI-neo Mammalian ExpressionVector; Promega (#E1841) harboring the point mutations T315I, F317L,F486S, and M351T; transfectants are selected for neomycin resistance andIL-3-independent growth (Weisberg et al., 2005). Ba/F3 cells are stablytransfected by electroporation with BCR-ABL point mutants identified ina random mutagenesis screen for BCR-ABL point mutants conferringresistance to nilotinib: Q252H, E292K, Y253C, Y253H, E255K, and V289L(Ray et al., 2007). Ba/F3 cells are made to express Tel-PDGFRβ asdescribed by Golub et al., 1994, and Carroll et al., 1996. Constructs ofD842V-PDGFRα and V561D-PDGFRα cDNA cloned into pcDNA3.1 are stablytransfected into Ba/F3 cells by electroporation, and cells are selectedfor neomycin resistance and IL3-independent growth as described byWeisberg et al., 2006.

All cell lines are cultured with 5% CO₂ at 37° C. in RPMI (Mediatech,Inc., Herndon, Va.) with 10% fetal calf serum (FCS) and supplementedwith 1% L-glutamine. Parental Ba/F3 cells are similarly cultured with15% WEHI-conditioned medium as a source of IL-3. Transfected cell linesare cultured in media supplemented with 1 mg/ml G418.

Antibodies and Immunoblotting and Immunoprecipitation

Anti-p-Tyr (clone 4G10, Upstate Biotechnology, NY) is used at 1:1000 forimmunoblotting. The ABL antibody (clone 24-21, Calbiochem, San Diego,Calif.) is used at 1:1000 for immunoblotting. The KIT antibody (C-19,Santa Cruz Biotechnology, CA) is used at 1:1000 for immunoblotting. Thephospho-KIT antibody (Tyr719, Cell Signaling, Danvers, Mass.) is used at1:1000 for immunoblotting. PDGFRA antibody (C-20, Santa CruzBiotechnology, CA) is used at 1:200 for immunoblotting. The monoclonalanti-β-actin antibody (clone AC-15) (Sigma-Aldrich, St. Louis, Mo.) isused at a 1:2000 dilution. Cells are lysed in lysis buffer (0.02 M Tris[pH 8.0], 0.15 M NaCl, 10% glycerol, 1% NP-40 (wt/vol), 0.1 M NaF, 1 mMphenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 40 μg/mlleupeptin, and 20 μg/ml aprotinin). Protein lysates are incubated for 25min on ice, with vortexing at 5 min intervals, and then centrifuged for15 min at 12,000×g. Supernatants are saved, and the Bio-Rad ProteinAssay is used to determine protein yields (Bio-Rad Laboratories,Hercules, Calif.). Equivalent amounts of protein are subsequently loadeddirectly onto a gel for immunoblotting experiments. Forimmunoprecipitation, cell lysates are incubated with FLT3/Flk-2 (C-20)antibody and protein A Sepharose overnight with rocking at 4° C. As acontrol, cell lysates are also incubated with protein A Sepharose beadsalone. Following incubation, immune complexes are washed 2× with lysisbuffer, 2× with 1×PBS, and are dissolved in Laemmeli's sample buffer byboiling for 5 min. For immunoblotting and immunoprecipitation, wholecell lysates and immune complexes, respectively, are resolved on asodium dodecyl sulfate (SDS)-7.5% polyacrylamide gel. Following this,protein is electrophoretically transferred to a Protran nitrocellulosetransfer and immobilization membrane (Schleicher and Schuell, Dassel,Germany). The membrane is then blocked overnight at 4° C. with 5% nonfatdry milk in 1×TBS (10 mM Tris-HCl [pH 8.0], 150 mM NaCl) and then probedfor 2 hr at 25° C. with pTYR, antibody or overnight at 4 C withFLT3/Flk-2 (C-20) antibody in 1×TBST buffer (10 mM Tris-HCl [pH 8.0],150 mM NaCl, 0.05% Tween20). Following 3 washes with 1×TBST, membranesare incubated for 1 hr at 25° C. with anti-mouse immunoglobulin(horseradish peroxidase-linked whole antibody from sheep) or anti-rabbitimmunoglobulin (horseradish peroxidase linked whole antibody fromdonkey) (Amersham Life Science, Inc., Arlington Heights, Ill.). Themembrane is washed 5× in 1×TBST buffer, with 5 min intervals betweenbuffer changes, and bound antibodies are detected with enhanced luminoland oxidizing reagent as specified by the manufacturer (NEN Life ScienceProducts, Boston, Mass.). Bound antibodies are removed with strippingbuffer (2% SDS, 0.0625 mol/L Tris [pH 6.8], and 0.7% 2-mercaptoethanol)50° C. for 30 min. The filter is then probed with additional antibodies.

Cell Cycle Analysis

Cell cycle analysis is performed using approximately 500,000 cells,which are centrifuged at 1500 rpm for 5 min, washed in PBS, and thepellet re-suspended in 500 μl of propidium iodide solution (50 μg/mlpropidium iodide, 0.1% NP-40, 0.1% sodium citrate). The mixture isstored in the dark at 4° C. for a minimum of 15 min, and then analyzedby flow cytometry. Apoptosis of drug-treated cells is measured using theAnnexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis).

Apoptosis Assay

Cells cultured in the presence or absence of drug are washed 1× withphosphate-buffered saline (PBS) and centrifuged for 5 min at 1500 rpm.Washed cell pellets are re-suspended in 100 μl of 20%Annexin-V-fluorescein labeling reagent and 20% propidium iodide (PI) inHEPES buffer. Cells are incubated for 15 min at room temperature,followed by dilution with 0.8 ml of HEPES buffer. Samples are thenanalyzed by flow cytometry. As controls, cells are incubated for 15 minwith PI alone, Annexin-V-fluorescein labeling reagent alone, or HEPESbuffer, and then diluted with HEPES buffer and analyzed by flowcytometry.

Drug Combination Studies

For drug combination studies, compounds are added simultaneously atfixed ratios to cells, and cell viability is determined by trypan blueexclusion and expressed as the function of growth affected (FA)drug-treated versus control cells. Synergy is assessed by Calcusynsoftware (Biosoft, Ferguson, Mo. and Cambridge, UK), using theChou-Talalay method (Chou and Talalay, 1984). The combinationindex=[D]₁[D_(x)]₁+[D]₂/[D_(x)]₂, where [D]₁ and [D]₂ are theconcentrations required by each drug in combination to achieve the sameeffect as concentrations [D_(x)]₁ and [D_(x)]₂ of each drug alone.Values less than one indicate synergy, whereas values greater than oneindicate antagonism.

Bioluminescent Imaging

Cells are transduced with a retrovirus encoding firefly luciferase(MSCV-Luc), and selected with puromycin at 2 μg/ml to generate 32D.p210-luc+ cells, as described by Weisberg et al., 2005. Ba/F3-KIT-T670Icells are transduced with MSCV-luc-neo vector following the sameprotocol as described by Weisberg et al., 2005.

Kinase Screen

KINOMEscan™ (Ambit Biosciences, San Diego, Calif.), a high-throughputmethod for screening small molecular agents against a large panel ofhuman kinases, is utilized. The technology is a competition bindingassay that profiles the selectivity of compounds against 350 kinases,each fused to a proprietary tag. The quantity of each kinase bound to animmobilized, active site-directed ligand is measured in the presence andabsence of compound.

Tables and Figures

TABLE I IC₅₀ values (in units of micromolar) of compounds against kinasetargets. Compound T3151 Bcr-Abl EML4-Alk Tel-Alk 1 — >10 >10 >10 2 0.630.05 4.19 4.48 3 — 0.47 1.67 1.07 6 0.31 3.07 1.34 2.907 >10 >10 >10 >10 8 >10 >10 >10 >10 9 >10 >10 >10 >10 10 >10 >10 >10 >1011 >10 >10 >10 >10 12 2.08 1.90 1.05 0.72 13 0.50 0.03 1.06 0.31 14 >101.05 5.60 1.44 15 >10 1.19 8.47 1.29 16 >10 0.22 >10 >10 17 7.120.78 >10 >10 18 3.08 1.57 >10 >10 19 0.34 0.65 0.06 0.03 20 0.31 0.090.40 0.27 21 0.24 0.53 0.36 0.32 22 1.27 0.36 0.50 0.4323 >10 >10 >10 >10 24 3.11 2.38 2.53 3.75 25 >10 7.18 >10 7.18 26 —7.08 >10 7.08 27 1.56 0.07 4.22 5.26 28 >10 1.44 >10 >10 29 >101.66 >10 >10 31 >10 7.96 7.09 8.35 32 >10 4.01 >10 >10

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

The invention claimed is:
 1. A compound of the Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is a bond;R^(1a) is selected from methyl, C(O)cyclopropyl, and C(O)-phenyl;wherein the C(O)-phenyl can be substituted with -piperazinyl-CH₃; R^(1b)is H; R^(A) is selected from H, methyl, and QR⁵, provided that oneinstance of R^(A) is QR⁵; wherein QR⁵ is selected from —C(O)—C₁₋₆alkyloxy, —C(O)N(H)—C₁₋₆ alkyl, —C(O)N(H)-phenyl, —NHC(O)—C₃₋₆cycloalkyl, —NHC(O)-phenyl, and —N(H)C(O)N(H)-phenyl; wherein the phenylring is optionally substituted with one to two substituents wherein thesubstituents are selected from one each of CF₃,—CH₂-piperazinyl-C₁₋₆alkyl, and -imidazolyl-C₁₋₆alkyl; and n is 1 or 2.2. The compound of claim 1 or a pharmaceutically acceptable saltthereof, wherein: QR⁵ is selected from:


3. A compound of formula (II), or a pharmaceutically acceptable saltthereof,

wherein: X is a bond; R^(1a) is selected from H, C₁₋₆ alkyl, C(O)—C₃₋₆cycloalkyl, C(O)-phenyl and C(O)-phenyl-piperazinyl-C₁₋₆ alkyl; R^(1b)is H; R² is selected from H and C₁₋₆ alkyl; R³ is H; R⁴ is QR⁵, whereinQ is selected from —C(O)N(H)—, —NHC(O)— and —N(H)C(O)N(H)—; R⁵ is:

wherein R⁶ is H; R⁷ is selected from H and C₁₋₆ trihaloalkyl; R⁸ isselected from H and —C₁₋₆ alkyl-heterocyclyl-C₁₋₆ alkyl; and R⁹ isselected from H and -heteroaryl-C₁₋₆ alkyl.
 4. The compound of claim 3,or a pharmaceutically acceptable salt thereof, wherein: R^(1a) is H,CH₃, C(O)-cyclopropyl, C(O)Ph or C(O)Ph-piperazinyl-CH₃; R² is H or CH₃;R⁷ is selected from H and CF₃; R⁸ is selected from H,—CH₂-piperazinyl-CH₃ and —CH₂-piperazinyl-CH₂CH₃; and R⁹ is selectedfrom H and -imidazolyl-CH₃.
 5. The compound of claim 3, or apharmaceutically acceptable salt thereof, wherein: R^(1a) is selectedfrom H and C(O)-cyclopropyl; R⁷ is CF₃; R⁸ is selected from H and1-ethyl-4-methylpiperazinyl; R⁹ is selected from H and4-methyl-1H-imidazolyl.
 6. The compound of claim 5, or apharmaceutically acceptable salt thereof, wherein: R^(1a) isC(O)-cyclopropyl.
 7. The compound of claim 3, or a pharmaceuticallyacceptable salt thereof, wherein: R^(1a) is selected from H, C₁₋₆ alkyl,C(O)—C₃₋₆ cycloalkyl, and C(O)-phenyl; and R⁴ is C(O)N(H)—R⁵; wherein R⁵is:

wherein R⁸ is 1-ethyl-4-methylpiperazinyl and R⁹ is H; or R⁴ isC(O)N(H)—R⁵; wherein R⁵ is:

wherein R⁸ is H and R⁹ is 4-methyl-1H-imidazolyl; or R⁴ is N(H)C(O)—R⁵;wherein R⁵ is:

wherein R⁸ is 1-ethyl-4-methylpiperazinyl and R⁹ is H.
 8. A compoundselected from

or a pharmaceutically acceptable salt thereof.
 9. A method of treatingcancer, comprising administering to a subject in need thereof a compoundof claim 1 or a pharmaceutically acceptable salt thereof.
 10. The methodof claim 9, wherein the cancer is selected from the group consisting ofadenocarcinoma, multiple myeloma, chronic myelogenous leukemia,pancreatic cancer, non-small cell lung cancer, lung cancer, breastcancer, colon cancer, ovarian cancer, cervical cancer, uterine cancer,prostate cancer, malignant melanoma, non-melanoma skin cancers,gastrointestinal stromal tumors, hematologic tumors, hematologicmalignancies, childhood leukemia, childhood lymphomas, multiple myeloma,Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas ofcutaneous origin, acute leukemia, chronic leukemia, acute lymphoblasticleukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasmacell neoplasm, lymphoid neoplasm and cancers associated with AIDS. 11.The method of claim 9, wherein the cancer is resistant to treatment withimatinib.
 12. The method of claim 9 further comprising administering apharmaceutically effective amount of an additional anti-cancer agent.13. The method of claim 12 wherein the additional anti-cancer agent isimatinib or nilotinib.
 14. A pharmaceutical composition comprising acompound of claim 1 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 15. A compound of the Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is NR; whereinR is selected from H and C₁₋₆ alkyl; R^(1a) is C(O)cyclopropyl; R^(1b)is H; R^(A) is selected from H, methyl, methoxy, and QR⁵, provided thatone instance of R^(A) is QR⁵; wherein QR⁵ is C(O)N(H)-phenyl, whereinthe phenyl ring is substituted with one or two substituents—selectedfrom one each of CF₃ and —CH₂-piperazinyl-C₁₋₆alkyl; and n is 1 or 2.16. The compound of claim 15 or a pharmaceutically acceptable saltthereof, wherein: R is H; and QR⁵ is selected from:


17. A compound of formula (II), or a pharmaceutically acceptable saltthereof,

wherein: X is NR; wherein R is selected from H and C₁₋₆ alkyl; R^(1a) isC(O)—C₃₋₆ cycloalkyl; R^(1b) is H; R² is selected from H and C₁₋₆ alkyl;R³ is H; R⁴ is QR⁵, wherein Q is —C(O)N(H)—; R⁵ is:

wherein R⁶ is H; R⁷ is selected from H and C₁₋₆ trihaloalkyl; R⁸ isselected from H and —C₁₋₆ alkyl-heterocyclyl-C₁₋₆ alkyl; and R⁹ is H.18. The compound of claim 17, or a pharmaceutically acceptable saltthereof, wherein: R is H; R^(1a) is C(O)-cyclopropyl; R² is H or CH₃; R⁷is selected from H and CF₃; and R⁸ is selected from H,—CH₂-piperazinyl-CH₃ and —CH₂-piperazinyl-CH₂CH₃.
 19. The compound ofclaim 18, or a pharmaceutically acceptable salt thereof, wherein R⁷ isCF₃.
 20. The compound of claim 18, or a pharmaceutically acceptable saltthereof, wherein R⁸ is —CH₂-piperazinyl-CH₃ or —CH₂-piperazinyl-CH₂CH₃.21. The compound of claim 15, or a pharmaceutically acceptable saltthereof, wherein R⁷ is CF₃.
 22. The compound of claim 20, wherein thecompound of Formula I is selected from

or a pharmaceutically acceptable salt thereof.
 23. A pharmaceuticalcomposition comprising a compound of claim 15 or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier. 24.A method of treating cancer, comprising administering to a subject inneed thereof a compound of claim 15 or a pharmaceutically acceptablesalt thereof.
 25. The method of claim 24, wherein the cancer is selectedfrom the group consisting of adenocarcinoma, multiple myeloma, chronicmyelogenous leukemia, pancreatic cancer, non-small cell lung cancer,lung cancer, breast cancer, colon cancer, ovarian cancer, cervicalcancer, uterine cancer, prostate cancer, malignant melanoma,non-melanoma skin cancers, gastrointestinal stromal tumors, hematologictumors, hematologic malignancies, childhood leukemia, childhoodlymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocyticorigin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia,acute lymphoblastic leukemia, acute myelocytic leukemia, chronicmyelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancersassociated with AIDS.
 26. The method of claim 24, wherein the cancer isresistant to treatment with imatinib.
 27. The method of claim 24 furthercomprising administering a pharmaceutically effective amount of anadditional anti-cancer agent.
 28. The method of claim 27 wherein theadditional anti-cancer agent is imatinib or nilotinib.